Tetrazolones as inhibitors of fatty acid synthase

ABSTRACT

Provided herein are tetrazolone FASN inhibitors of the formula (I): 
     
       
         
         
             
             
         
       
     
     or a pharmaceutically acceptable form thereof, wherein the variables R A , R B  and R C  are defined herein. 
     Also provided herein are pharmaceutical compositions of the compounds provided herein as well as methods of their use for the treatment of various disorders such as hyperproliferative disorders, inflammatory disorders, obesity-related disorders and microbial infections.

This application claims priority to U.S. Provisional Application Nos.61/331,575, filed May 5, 2010; 61/331,644, filed May 5, 2010;61/437,564, filed Jan. 28, 2011; and 61/472,566, filed Apr. 6, 2011, allof which are incorporated herein in their entireties.

A text file of sequence listing (SEQLIST 12928-033-999.TXT; careated May4, 2011; size 20,000 bytes), filed electronically, is incorporatedherein by reference.

BACKGROUND

Fatty acid synthase (FASN) is a key enzyme for the synthesis oflong-chain fatty acids from acetyl-coenzyme A (CoA) and malonyl-CoA thatuses reduced nicotinamide adenine dinucleotidephosphate as a cofactor.FASN is minimally expressed in most normal human tissues except theliver and adipose tissue, where it is expressed at high levels. SinceFASN expression is markedly increased in several human cancers comparedwith the corresponding normal tissue, and FASN overexpression in tumorshas been associated with a poor prognosis, FASN inhibitors have longbeen viewed as potential therapeutics for the treatment of cancer. FASNinhibitors have also shown promise in the treatment of otherFASN-mediated diseases, disorders or conditions, such as obesity, lackof appetite control and inflammatory conditions.

Furthermore, FASN has been identified as a target for treatment ofmicrobial infections. In particular, it was reported that fatty acidsynthesis or the level of fatty acid is critical in viral pathogenesis.For example, it was reported that the formation of a novel vesicularcompartment (i.e., remodelled golgi apparatus), on the surface of whichviral RNA replication takes place, requires fatty acid biosynthesis.(See Cherry et al., PLoS Pathogens, 2(10): e102 (2006)). In addition,fatty acid biosynthesis has been indentified as a target for anti-viraltherapy using a metabolic profiling of the hosts upon viral infection.(See Munger et al., Nature Biotechnology, 26: 1179-1186 (2008). It wasalso reported that inhibition of fatty acid biosynthesis (e.g.,inhibition of fatty acid synthase) results in reduced replication ofhuman cytomegalomous virus (HCMV) and influenza A viruses. (Id.).

Reports establishing FASN as a valid target for the treatment of viralinfections are available for various viruses. For example, the role ofFASN has been implicated in the pathogenesis of an enveloped virus suchas human cytomegalomous virus (HCMV), influenza A and Hepatitis C (HCV).(See Munger et al., Nature Biotechnology, 26: 1179-1186 (2008); Syed etal., Trends in Endocrinology and Metabolism, 21: 33-40 (2009); Sakamotoet al., Nature Chemcial Biology, 1: 333-337 (2005); Yang et al.,Hepatology, 48: 1396-1403 (2008)). With regard to HCV, it was reportedthat an elevated level of fatty acid biosynthesis enzymes, includingFASN, contributes to liver steatosis, leading to cirrhosis andhepatocellular carcinoma, upon HCV infection. (Fukusawa et al., Biol.Pharm. Bull., 29(9): 1958-1961 (2006)). HCV replication was reported tobe regulated by, among others, fatty acid biosynthesis. (Kapadia et al.,Proc. Natl. Acad. Sci., 102(7): 2561-2566 (2005)). Other reportsestablishing FASN as a potential host-target against HCV have also beenpublished. (See, e.g., Hepatology, 48: 1396 (2008); Trends EndocrineMetabol., 21: 33 (2010); and Virology, 394: 130 (2009)).

With regard to other various viruses, it was reported that the FASNexpression is increased in the cells infected by coxsackievirus B3(CVB3), a picornavirus, and the replication of CVB3 is blocked by FASNinhibitors. (See Rassmann et al., Antiviral Research, 76:150-158(2007)). FASN was reported to be important in lytic viralreplication of Epstein-Barr virus (EBV), and it was suggested that FASNinhibition may be a novel approach for blocking the EBV replication. (Liet al., Journal of Virology, 78(8): 4197-4206 (2004)). The role of FASNin the replication of dengue virus has also been implicated. (See, e.g.,Heaton et al., Proc. Natl. Acad. Sci., 107(40): 17345-17350 (2010); andSamsa et al., PLoS Pathegens, 5(10): e1000632 (2009)). Moreover, asidefrom being a potential target for anti-viral therapy, the role of FASNhas also been implicated in diabetes or regulation of the generalwellness of the liver. (See, e.g., Wu et al., PNAS Early Edition,www.pnas.org/cgi/doi/10.1073/pnas.1002588108 (2011)). Thus, there is aneed for effective inhibitors of FASN, which can be potentially used astherpies for microbial infections, including, but not limited to viralinfections, or other diseases and disorders.

SUMMARY

Provided herein are tetrazolone FASN inhibitors of the formula (I):

or a pharmaceutically acceptable form thereof; wherein the variablesR^(A), R^(B) and R^(C) are defined below and herein.

Also provided herein are pharmaceutical compositions comprising at leastone compound of formula (I) or a pharmaceutically acceptable formthereof. Also provided herein are methods of treating cancer comprisingadministering at least one compound of formula (I) or a pharmaceuticallyacceptable form thereof, or a pharmaceutical composition thereof, to asubject in need thereof. Also provided herein are methods of treatingmicrobial infections comprising administering at least one compound offormula (I) or a pharmaceutically acceptable form thereof, or apharmaceutical composition thereof, to a subject in need thereof.

The details of additional or alternative embodiments are set forth inthe accompanying Detailed Description and Exemplification as describedbelow. Other features, objects, and advantages will be apparent fromthis description and from the claims.

SEQUENCE IDENTIFICATION NUMBERS

SEQ ID NO. 1: Homo sapiens FASN amino acid sequence:MEEVVIAGMSGKLPESENLQEFWDNLIGGVDMVTDDDRRWKAGLYGLPRRSGKLKDLSRFDASFFGVHPKQAHTMDPQLRLLLEVTYEAIVDGGINPDSLRGTHTGVWVGVSGSETSEALSRDPETLVGYSMVGCQRAMMANRLSFFFDFRGPSIALDTACSSSLMALQNAYQAIHSGQCPAAIVGGINVLLKPNTSVQFLRLGMLSPEGTCKAFDTAGNGYCRSEGVVAVLLTKKSLARRVYATILNAGTNTDGFKEQGVTFPSGDIQEQLIRSLYQSAGVAPESFEYIEAHGTGTKVGDPQELNGITRALCATRQEPLLIGSTKSNMGHPEPASGLAALAKVLLSLEHGLWAPNLHFHSPNPEIPALLDGRLQVVDQPLPVRGGNVGINSFGFGGSNVHIILRPNTQPPPAPAPHATLPRLLRASGRTPEAVQKLLEQGLRHSQDLAFLSMLNDIAAVPATAMPFRGYAVLGGERGGPEVQQVPAGERPLWFICSGMGTQWRGMGLSLMRLDRFRDSILRSDEAVKPFGLKVSQLLLSTDESTFDDIVHSFVSLTAIQIGLIDLLSCMGLRPDGIVGHSLGEVACGYADGCLSQEEAVLAAYWRGQCIKEAHLPPGAMAAVGLSWEECKQRCPPGVVPACHNSKDTVTISGPQAPVFEFVEQLRKEGVFAKEVRTGGMAFHSYFMEAIAPPLLQELKKVIREPKPRSARWLSTSIPEAQWHSSLARTSSAEYNVNNLVSPVLFQEALWHVPEHAVVLEIAPHALLQAVLKRGLKPSCTIIPLMKKDHRDNLEFFLAGIGRLHLSGIDANPNALFPPVEFPAPRGTPLISPLIKWDHSLAWDVPAAEDFPNGSGSPSAAIYNIDTSSESPDHYLVDHTLDGRVLFPATGYLSIVWKTLARALGLGVEQLPVVFEDVVLHQATILPKTGTVSLEVRLLEASRAFEVSENGNLVVSGKVYQWDDPDPRLFDHPESPTPNPTEPLFLAQAEVYKELRLRGYDYGPHFQGILEASLEGDSGRLLWKDNWVSFMDTMLQMSILGSAKHGLYLPTRVTAIHIDPATHRQKLYTLQDKAQVADVVVSRWLRVTVAGGVHISGLHTESAPRRQQEQQVPILEKFCFTPHTEEGCLSERAALQEELQLCKGLVQALQTKVTQQGLKMVVPGLDGAQIPRDPSQQELPRLLSAACRLQLNGNLQLELAQVLAQERPKLPEDPLLSGLLDSPALKACLDTAVENMPSLKMKVVEVLAGHGHLYSRIPGLLSPHPLLQLSYTATDRHPQALEAAQAELQQHDVAQGQWDPADPAPSALGSADLLVCNCAVAALGDPASALSNMVAALREGGFLLLHTLLRGHPLGDIVAFLTSTEPQYGQGILSQDAWESLFSRVSLRLVGLKKSFYGSTLFLCRRPTPQDSPIFLPVDDTSFRWVESLKGILADEDSSRPVWLKAINCATSGVVGLVNCLRREPGGNRLRCVLLSNLSSTSHVPEVDPGSAELQKVLQGDLVMNVYRDGAWGAFRHFLLEEDKPEEPTAHAFVSTLTRGDLSSIRWVCSSLRHAQPTCPGAQLCTVYYASLNFRDIMLATGKLSPDAIPGKWTSQDSLLGMEFSGRDASGKRVMGLVPAKGLATSVLLSPDFLWDVPSNWTLEEAASVPVVYSTAYYALVVRGRVRPGETLLIHSGSGGVGQAAIAIALSLGCRVFTTVGSAEKRAYLQARFPQLDSTSFANSRDTSFEQHVLWHTGGKGVDLVLNSLAEEKLQASVRCLATHGRFLEIGKFDLSQNHPLGMAIFLKNVTFHGVLLDAFFNESSADWREVWALVQAGIRDGVVRPLKCTVFHGAQVEDAFRYMAQGKHIGKVVVQVLAEEPEAVLKGAKPKLMSAISKTFCPAHKSYIIAGGLGGFGLELAQWLIQRGVQKLVLTSRSGIRTGYQAKQVRRWRRQGVQVQVSTSNISSLEGARGLIAEAAQLGPVGGVFNLAVVLRDGLLENQTPEFFQDVCKPKYSGTLNLDRVTREACPELDYFVVFSSVSCGRGNAGQSNYGFANSAMERICEKRRHEGLPGLAVQWGAIGDVGILVETMSTNDTIVSGTLPQRMASCLEVLDLFLNQPHMVLSSFVLAEKAAAYRDRDSQRDLVEAVAHILGIRDLAAVNLDSSLADLGLDSLMSVEVRQTLERELNLVLSVREVRQLTLRKLQELSSKADEASELACPTPKEDGLAQQQTQLNLRSLLVNPEGPTLMRLNSVQSSERPLFLVHPIEGSTTVFHSLASRLSIPTYGLQCTRAAPLDSIHSLAAYYIDCIRQVQPEGPYRVAGYSYGACVAFEMCSQLQAQQSPAPTHNSLFLFDGSPTYVLAYTQSYRAKLTPGCEAEAETEAICFFVQQFTDMEHNRVLEALLPLKGLEERVAAAVDLIIKSHQGLDRQELSFAARSFYYKLRAAEQYTPKAKYHGNVMLLRAKTGGAYGEDLGADYNLSQVCDGKVSVHVIEGDHRTLLEGSGLESIISIIHSS LAEPRVSVREG

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March March's Advanced Organic Chemistry, 5^(th) Edition, JohnWiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Certain compounds provided herein can comprise one or more asymmetriccenters, and thus can exist in various isomeric forms, e.g., enantiomersand/or diastereomers and/or stereoisomers. The compounds provided hereincan be in the form of an individual enantiomer, diastereomer orgeometric isomer, or can be in the form of a mixture of stereoisomers,including racemic mixtures and mixtures enriched in one or morestereoisomer. In certain embodiments, the compounds provided herein areenantiopure compounds. In certain other embodiments, mixtures ofstereoisomers are provided.

Furthermore, certain compounds, as described herein can have one or moredouble bonds that can exist as either the cis or trans, or the E or Zisomer, unless otherwise indicated. Also encompassed are the compoundsas individual isomers substantially free of other isomers, andalternatively, as mixtures of various isomers, e.g., racemic mixtures ofE/Z isomers or mixtures enriched in one E/Z isomer.

The terms “optically enriched”, “enantiomerically enriched,”“enantiomerically pure” and “non-racemic,” as used interchangeablyherein, refer to compositions in which the percent by weight of oneenantiomer is greater than the amount of that one enantiomer in acontrol mixture of the racemic composition (e.g., greater than 1:1 byweight). In addition, the term “non-racemic” can apply more broadly tomixtures of stereoisomers, diastereomers or olefin E/Z isomers. Forexample, an enantiomerically enriched preparation of the (S)-enantiomer,means a preparation of the compound having greater than 50% by weight ofthe (S)-enantiomer relative to the (R)-enantiomer, such as at least 75%by weight, and even such as at least 80% by weight. In some embodiments,the enrichment can be much greater than 80% by weight, providing a“substantially optically enriched,” “substantially enantiomericallyenriched,” “substantially enantiomerically pure” or a “substantiallynon-racemic” preparation, which refers to preparations of compositionswhich have at least 85% by weight of one enantiomer relative to otherenantiomer, such as at least 90% by weight, and such as at least 95% byweight. In some embodiments, the enantiomerically enriched compositionhas a higher potency with respect to therapeutic utility per unit massthan does the racemic mixture of that composition. Enantiomers can beisolated from mixtures by methods known to those skilled in the art,including chiral high pressure liquid chromatography (HPLC) and theformation and crystallization of chiral salts; or enantiomers can beprepared by asymmetric syntheses. See, for example, Jacques, et al.,Enantiomers, Racemates and Resolutions (Wiley Interscience, New York,1981); Wilen, S. H., et al., Tetrahedron 33:2725 (1977); Eliel, E. L.Stereochemistry of Carbon Compounds (McGraw-Hill, N Y, 1962); and Wilen,S. H. Tables of Resolving Agents and Optical Resolutions p. 268 (E. L.Eliel, Ed., Univ. of Notre Dame Press, Notre Dame, Ind. 1972).

As used herein, alone or as part of another group, “halo” and “halogen”refer to fluorine (fluoro, —F), chlorine (chloro, —Cl), bromine (bromo,—Br), or iodine (iodo, —I).

As used herein, alone or as part of another group, “alkyl” refers to amonoradical of a straight-chain or branched saturated hydrocarbon grouphaving from 1 to 10 carbon atoms (“C₁₋₁₀ alkyl”). In some embodiments,an alkyl group has 1 to 9 carbon atoms (“C₁₋₉ alkyl”). In someembodiments, an alkyl group has 1 to 8 carbon atoms (“C₁₋₈ alkyl”). Insome embodiments, an alkyl group has 1 to 7 carbon atoms (“C₁₋₇ alkyl”).In some embodiments, an alkyl group has 1 to 6 carbon atoms (“C₁₋₆alkyl”). In some embodiments, an alkyl group has 1 to 5 carbon atoms(“C₁₋₅ alkyl”). In some embodiments, an alkyl group has 1 to 4 carbonatoms (“C₁₋₄ alkyl”). In some embodiments, an alkyl group has 1 to 3carbon atoms (“C₁₋₃ alkyl”). In some embodiments, an alkyl group has 1to 2 carbon atoms (“C₁₋₂ alkyl”). In some embodiments, an alkyl grouphas 1 carbon atom (“C₁ alkyl”). In some embodiments, an alkyl group has2 to 6 carbon atoms (“C₂₋₆ alkyl”). Examples of C₁₋₆ alkyl groupsinclude methyl (C₁), ethyl (C₂), n-propyl (C₃), isopropyl (C₃), n-butyl(C₄), tert-butyl (C₄), sec-butyl (C₄), iso-butyl (C₄), n-pentyl (C₅),3-pentanyl (C₅), amyl (C₅), neopentyl (C₅), 3-methyl-2-butanyl (C₅),tertiary amyl (C₅), and n-hexyl (C₆). Additional examples of alkylgroups include n-heptyl (C₇), n-octyl (C₈) and the like. Unlessotherwise specified, each instance of an alkyl group is independentlyunsubstituted (an “unsubstituted alkyl”) or substituted (a “substitutedalkyl”) with 1, 2, 3, 4, or 5 substituents as described herein. Incertain embodiments, the alkyl group is an unsubstituted C₁₋₁₀ alkyl(e.g., —CH₃). In certain embodiments, the alkyl group is a substitutedC₁₋₁₀ alkyl.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

“Perhaloalkyl” as defined herein refers to an alkyl group having from 1to 10 carbon atoms wherein all of the hydrogen atoms are eachindependently replaced by a halogen, e.g., selected from fluoro, bromo,chloro or iodo (“C₁₋₁₀ perhaloalkyl”). In some embodiments, the alkylmoiety has 1 to 9 carbon atoms (“C₁₋₉ perhaloalkyl”). In someembodiments, the alkyl moiety has 1 to 8 carbon atoms (“C₁₋₈perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 7 carbonatoms (“C₁₋₇ perhaloalkyl”). In some embodiments, the alkyl moiety has 1to 6 carbon atoms (“C₁₋₆ perhaloalkyl”). In some embodiments, the alkylmoiety has 1 to 5 carbon atoms (“C₁₋₅ perhaloalkyl”). In someembodiments, the alkyl moiety has 1 to 4 carbon atoms (“C₁₋₄perhaloalkyl”). In some embodiments, the alkyl moiety has 1 to 3 carbonatoms (“C₁₋₃ perhaloalkyl”). In some embodiments, the alkyl moiety has 1to 2 carbon atoms (“C₁₋₂ perhaloalkyl”). In some embodiments, all of thehydrogen atoms are each replaced with fluoro. In some embodiments, allof the hydrogen atoms are each replaced with chloro. Examples ofperhaloalkyl groups include —CF₃, —CF₂CF₃, —CF₂CF₂CF₃, —CCl₃, —CFCl₂,—CF₂Cl and the like.

As used herein, alone or as part of another group, “alkenyl” refers to amonoradical of a straight-chain or branched hydrocarbon group havingfrom 2 to 10 carbon atoms and one or more carbon-carbon double bonds(“C₂₋₁₀ alkenyl”). In some embodiments, an alkenyl group has 2 to 9carbon atoms (“C₂₋₉ alkenyl”). In some embodiments, an alkenyl group has2 to 8 carbon atoms (“C₂₋₈ alkenyl”). In some embodiments, an alkenylgroup has 2 to 7 carbon atoms (“C₂₋₇ alkenyl”). In some embodiments, analkenyl group has 2 to 6 carbon atoms (“C₂-alkenyl”). In someembodiments, an alkenyl group has 2 to 5 carbon atoms (“C₂₋₅ alkenyl”).In some embodiments, an alkenyl group has 2 to 4 carbon atoms (“C₂₋₄alkenyl”). In some embodiments, an alkenyl group has 2 to 3 carbon atoms(“C₂₋₃ alkenyl”). In some embodiments, an alkenyl group has 2 carbonatoms (“C₂ alkenyl”). The one or more carbon-carbon double bonds can beinternal (such as in 2-butenyl) or terminal (such as in 1-butenyl).Examples of C₂₋₄ alkenyl groups include ethenyl (C₂), 1-propenyl (C₃),2-propenyl (C₃), 1-butenyl (C₄), 2-butenyl (C₄), butadienyl (C₄) and thelike. Examples of C₂₋₆ alkenyl groups include the aforementioned C₂₋₄alkenyl groups as well as pentenyl (C₅), pentadienyl (C₅), hexenyl (C₆)and the like. Additional examples of alkenyl include heptenyl (C₇),octenyl (C₈), octatrienyl (C₈) and the like. Unless otherwise specified,each instance of an alkenyl group is independently unsubstituted (an“unsubstituted alkenyl”) or substituted (a “substituted alkenyl”) with1, 2, 3, 4, or 5 substituents as described herein. In certainembodiments, the alkenyl group is an unsubstituted C₂₋₁₀ alkenyl. Incertain embodiments, the alkenyl group is a substituted C₂₋₁₀ alkenyl.

As used herein, alone or as part of another group, “alkynyl” refers to amonoradical of a straight-chain or branched hydrocarbon group havingfrom 2 to 10 carbon atoms and one or more carbon-carbon triple bonds(“C₂₋₁₀ alkynyl”). In some embodiments, an alkynyl group has 2 to 9carbon atoms (“C₂₋₉ alkynyl”). In some embodiments, an alkynyl group has2 to 8 carbon atoms (“C₂₋₈ alkynyl”). In some embodiments, an alkynylgroup has 2 to 7 carbon atoms (“C₂₋₇ alkynyl”). In some embodiments, analkynyl group has 2 to 6 carbon atoms (“C₂₋₆ alkynyl”). In someembodiments, an alkynyl group has 2 to 5 carbon atoms (“C₂₋₅ alkynyl”).In some embodiments, an alkynyl group has 2 to 4 carbon atoms (“C₂₋₄alkynyl”). In some embodiments, an alkynyl group has 2 to 3 carbon atoms(“C₂₋₃ alkynyl”). In some embodiments, an alkynyl group has 2 carbonatoms (“C₂ alkynyl”). The one or more carbon-carbon triple bonds can beinternal (such as in 2-butynyl) or terminal (such as in 1-butynyl).Examples of C₂₋₄ alkynyl groups include, without limitation, ethynyl(C₂), 1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl (C₄), 2-butynyl (C₄)and the like. Examples of C₂₋₆ alkenyl groups include the aforementionedC₂₋₄ alkynyl groups as well as pentynyl (C₅), hexynyl (C₆) and the like.Additional examples of alkynyl include heptynyl (C₇), octynyl (C₈) andthe like. Unless otherwise specified, each instance of an alkynyl groupis independently unsubstituted (an “unsubstituted alkynyl”) orsubstituted (a “substituted alkynyl”) with 1, 2, 3, 4, or 5 substituentsas described herein. In certain embodiments, the alkynyl group is anunsubstituted C₂₋₁₀ alkynyl. In certain embodiments, the alkynyl groupis a substituted C₂₋₁₀ alkynyl.

As used herein, alone or as part of another group, “heteroaliphatic”refers to a monoradical of an acyclic 3- to 14-membered straight-chainor branched-chain having from 2 to 13 carbon atoms and 1 to 4heteroatoms selected from oxygen, sulfur, phosphorous, and nitrogen, andwherein the point of attachment is a carbon atom (“3-14 memberedheteroaliphatic”). In some embodiments, “heteroaliphatic” is a saturatedgroup (“heteroalkyl”). In some embodiments, “heteroaliphatic” is a groupcontaining one or more double bonds (“heteroalkenyl”). In someembodiments, “heteroaliphatic” is a group containing one or more triplebonds (“heteroalkynyl”). Exemplary heteroaliphatic groups include,without limitation, ethers such as methoxyethanyl (—CH₂CH₂OCH₃),ethoxymethanyl (—CH₂OCH₂CH₃), (methoxymethoxy)ethanyl (—CH₂CH₂OCH₂OCH₃),(methoxymethoxy)methanyl (—CH₂OCH₂OCH₃) and (methoxyethoxy)methanyl(—CH₂OCH₂CH₂OCH₃) and the like; amines such as —CH₂CH₂NHCH₃,—CH₂CH₂N(CH₃)₂, —CH₂NHCH₂CH₃, —CH₂N(CH₂CH₃)(CH₃) and the like. Unlessotherwise specified, each instance of a heteroaliphatic group isindependently unsubstituted (an “unsubstituted heteroaliphatic”) orsubstituted (a “substituted heteroaliphatic”) with 1-5 substituents asdescribed herein. In certain embodiments, the heteroaliphatic group isan unsubstituted 3-14 membered heteroaliphatic. In certain embodiments,the heteroaliphatic group is a substituted 3-14 memberedheteroaliphatic.

As used herein, alone or as part of another group, “carbocyclyl” refersto a radical of a non-aromatic cyclic hydrocarbon group having from 3 to10 ring carbon atoms (“C₃₋₁₀ carbocyclyl”) and zero heteroatoms in thenon-aromatic ring system. In some embodiments, a carbocyclyl group has 3to 9 ring carbon atoms (“C₃₋₉ carbocyclyl”). In some embodiments, acarbocyclyl group has 3 to 8 ring carbon atoms (“C₃₋₈ carbocyclyl”). Insome embodiments, a carbocyclyl group has 3 to 7 ring carbon atoms(“C₃₋₇ carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to6 ring carbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, acarbocyclyl group has 3 to 5 ring carbon atoms (“C₃₋₅ carbocyclyl”). Insome embodiments, a carbocyclyl group has 3 to 4 ring carbon atoms(“C₃₋₄ carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to10 ring carbon atoms (“C₅₋₁₀ carbocyclyl”). Examples of C₃₋₆ carbocyclylgroups include, without limitation, cyclopropyl (C₃), cyclobutyl (C₄),cyclopentyl (C₅), cyclopentenyl (C₅), cyclohexyl (C₆), cyclohexenyl(C₆), cyclohexadienyl (C₆) and the like. Examples of C₃₋₈ carbocyclylgroups include the aforementioned C₃₋₆ carbocyclyl groups as well ascycloheptyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), bicyclo[2.2.1]heptanyl, bicyclo[2.2.2]octanyl, and thelike. Examples of C₃₋₁₀ carbocyclyl groups include the aforementionedC₃₋₈ carbocyclyl groups as well as octahydro-1H-indenyl,decahydronaphthalenyl, spiro[4.5]decanyl and the like. As the foregoingexamples illustrate, in certain embodiments, the carbocyclyl group iseither monocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g.,containing a fused, bridged or spiro ring system such as a bicyclicsystem (“bicyclic carbocyclyl”) or tricyclic system (“tricycliccarbocyclyl”)) and can be saturated or can contain one or morecarbon-carbon double or triple bonds. “Carbocyclyl” also includes ringsystems wherein the carbocyclyl ring, as defined above, is fused withone or more aryl or heteroaryl groups wherein the point of attachment ison the carbocyclyl ring. Unless otherwise specified, each instance of acarbocyclyl group is independently unsubstituted (an “unsubstitutedcarbocyclyl”) or substituted (a “substituted carbocyclyl”) with 1, 2, 3,4, or 5 substituents as described herein. In certain embodiments, thecarbocyclyl group is an unsubstituted C₃₋₁₀ carbocyclyl. In certainembodiments, the carbocyclyl group is a substituted C₃₋₁₀ carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 10 ring carbon atoms (“C₃₋₁₀cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 8 ringcarbon atoms (“C₃₋₈ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 6 ring carbon atoms (“C₃₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 6 ring carbon atoms (“C₅₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ cycloalkyl”). Examples of C₅₋₆ cycloalkyl groupsinclude cyclopentyl (C₅) and cyclohexyl (C₅). Examples of C₃₋₆cycloalkyl groups include the aforementioned C₅₋₆ cycloalkyl groups aswell as cyclopropyl (C₃) and cyclobutyl (C₄). Examples of C₃₋₈cycloalkyl groups include the aforementioned C₃₋₆ cycloalkyl groups aswell as cycloheptyl (C₇) and cyclooctyl (C₈). Unless otherwisespecified, each instance of a cycloalkyl group is independentlyunsubstituted (an “unsubstituted cycloalkyl”) or substituted (a“substituted cycloalkyl”) with 1, 2, 3, 4, or 5 substituents asdescribed herein. In certain embodiments, the cycloalkyl group is anunsubstituted C₃₋₁₀ cycloalkyl. In certain embodiments, the cycloalkylgroup is a substituted C₃₋₁₀ cycloalkyl.

As used herein, alone or as part of another group, “heterocyclyl” refersto a radical of a 3- to 14-membered non-aromatic ring system having ringcarbon atoms and 1 to 4 ring heteroatoms, wherein each heteroatom isindependently selected from nitrogen, oxygen, phosphorous, and sulfur(“3-14 membered heterocyclyl”). In heterocyclyl groups that contain oneor more nitrogen or phosphorous atoms, the point of attachment can be acarbon, nitrogen, or phosphorous atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or polycyclic (e.g., a fused, bridged or spiro ring system such as abicyclic system (“bicyclic heterocyclyl”) or tricyclic system(“tricyclic heterocyclyl”)), and can be saturated or can contain one ormore carbon-carbon double or triple bonds. Heterocyclyl polycyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocycyl ring,as defined above, is fused with one or more carbocycyl groups whereinthe point of attachment is either on the carbocycyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring. In some embodiments, aheterocyclyl group is a 5-10 membered non-aromatic ring system havingring carbon atoms and 1-4 ring heteroatoms, wherein each heteroatom isindependently selected from nitrogen, oxygen, phosphorous, and sulfur(“5-10 membered heterocyclyl”). In some embodiments, a heterocyclylgroup is a 5-8 membered non-aromatic ring system having ring carbonatoms and 1-4 ring heteroatoms, wherein each heteroatom is independentlyselected from nitrogen, oxygen, phosphorous, and sulfur (“5-8 memberedheterocyclyl”). In some embodiments, a heterocyclyl group is a 5-6membered non-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, phosphorous, and sulfur (“5-6 membered heterocyclyl”).In some embodiments, the 5-6 membered heterocyclyl has 1-3 ringheteroatoms selected from nitrogen, oxygen, phosphorous, and sulfur. Insome embodiments, the 5-6 membered heterocyclyl has 1-2 ring heteroatomsselected from nitrogen, oxygen, phosphorous, and sulfur. In someembodiments, the 5-6 membered heterocyclyl has 1 ring heteroatomselected from nitrogen, oxygen, phosphorous, and sulfur. Exemplary3-membered heterocyclyls containing 1 heteroatom include, withoutlimitation, azirdinyl, oxiranyl, and thiorenyl. Exemplary 4-memberedheterocyclyls containing 1 heteroatom include, without limitation,azetidinyl, oxetanyl and thietanyl. Exemplary 5-membered heterocyclylscontaining 1 heteroatom include, without limitation, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl,dihydropyrrolyl and pyrrolyl-2,5-dione. Exemplary 5-memberedheterocyclyls containing 2 heteroatoms include, without limitation,dioxolanyl, oxathiolanyl and dithiolanyl. Exemplary 5-memberedheterocyclyls containing 3 heteroatoms include, without limitation,triazolinyl, oxadiazolinyl, and thiadiazolinyl. Exemplary 6-memberedheterocyclyl groups containing 1 heteroatom include, without limitation,piperidinyl, tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary6-membered heterocyclyl groups containing 2 heteroatoms include, withoutlimitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary6-membered heterocyclyl groups containing 2 heteroatoms include, withoutlimitation, triazinanyl. Exemplary 7-membered heterocyclyl groupscontaining 1 heteroatom include, without limitation, azepanyl, oxepanyland thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1heteroatom include, without limitation, azocanyl, oxecanyl andthiocanyl. Exemplary bicyclic heterocyclyl groups include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetra-hydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like. Unless otherwisespecified, each instance of heterocyclyl is independently unsubstituted(an “unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with 1, 2, 3, 4, or 5 substituents as described herein.In certain embodiments, the heterocyclyl group is an unsubstituted 3-14membered heterocyclyl. In certain embodiments, the heterocyclyl group isa substituted 3-14 membered heterocyclyl.

As used herein, alone or as part of another group, “aryl” refers to aradical of a monocyclic or polycyclic (e.g., bicyclic or tricyclic)aromatic ring system (e.g., having 6, 10 or 14 π electrons shared in acyclic array) having 6-14 ring carbon atoms and zero heteroatomsprovided in the aromatic ring system (“C₆₋₁₄ aryl”). In someembodiments, an aryl group has 6 ring carbon atoms (“C₆ aryl”; e.g.,phenyl). In some embodiments, an aryl group has 10 ring carbon atoms(“C₁₀ aryl”; e.g., naphthyl, such as 1-naphthyl and 2-naphthyl). In someembodiments, an aryl group has 14 ring carbon atoms (“C₁₄ aryl”; e.g.,anthracyl). “Aryl” also includes ring systems wherein the aryl ring, asdefined above, is fused with one or more carbocyclyl or heterocyclylgroups wherein the radical or point of attachment is on the aryl ring.Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with 1, 2, 3, 4, or 5 substituents as describedherein. In certain embodiments, the aryl group is an unsubstituted C₆₋₁₄aryl. In certain embodiments, the aryl group is a substituted C₆₋₁₄aryl.

As used herein, alone or part of another group, “aralkyl” refers to aC₁₋₁₀ alkyl group as defined herein substituted by a C₆₋₁₄ aryl group asdefined herein, wherein the point of attachment is on the alkyl group(“C₁₋₁₀ aralkyl”).

As used herein, alone or as part of another group, “heteroaryl” refersto a radical of a 5-14 membered monocyclic or polycyclic (e.g., bicyclicor tricyclic) aromatic ring system (e.g., having 6, 10 or 14 π electronsshared in a cyclic array) having ring carbon atoms and 1-4 ringheteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen, phosphorous,and sulfur (“5-14 membered heteroaryl”). In heteroaryl groups thatcontain one or more nitrogen or phosphorous atoms, the point ofattachment can be a carbon, phosphorous or nitrogen atom, as valencypermits. Heteroaryl polycyclic ring systems can include one or moreheteroatoms in one or both rings. “Heteroaryl” also includes ringsystems wherein the heteroaryl ring, as defined above, is fused with oneor more aryl groups wherein the point of attachment is either on thearyl or on the heteroaryl ring, or wherein the heteroaryl ring, asdefined above, is fused with one or more carbocycyl or heterocycylgroups wherein the point of attachment is on the heteroaryl ring. Forpolycyclic heteroaryl groups wherein one ring does not contain aheteroatom (e.g., indolyl, quinolinyl, carbazolyl and the like) thepoint of attachment can be on either ring, i.e., either the ring bearinga heteroatom (e.g., 2-indolyl) or the ring that does not contain aheteroatom (e.g., 5-indolyl). In some embodiments, a heteroaryl group isa 5-10 membered aromatic ring system having ring carbon atoms and 1-4ring heteroatoms provided in the aromatic ring system, wherein eachheteroatom is independently selected from nitrogen, oxygen, phosphorous,and sulfur (“5-10 membered heteroaryl”). In some embodiments, aheteroaryl group is a 5-8 membered aromatic ring system having ringcarbon atoms and 1-4 ring heteroatoms provided in the aromatic ringsystem, wherein each heteroatom is independently selected from nitrogen,oxygen, phosphorous, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, phosphorous, and sulfur (“5-6 memberedheteroaryl”). In some embodiments, the 5-6 membered heteroaryl has 1-3ring heteroatoms selected from nitrogen, oxygen, phosphorous, andsulfur. In some embodiments, the 5-6 membered heteroaryl has 1-2 ringheteroatoms selected from nitrogen, oxygen, phosphorous, and sulfur. Insome embodiments, the 5-6 membered heteroaryl has 1 ring heteroatomselected from nitrogen, oxygen, phosphorous, and sulfur. Exemplary5-membered heteroaryls containing 1 heteroatom include, withoutlimitation, pyrrolyl, furanyl and thiophenyl. Exemplary 5-memberedheteroaryls containing 2 heteroatoms include, without limitation,imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryls containing 3 heteroatomsinclude, without limitation, triazolyl, oxadiazolyl, and thiadiazolyl.Exemplary 5-membered heteroaryls containing 4 heteroatoms include,without limitation, tetrazolyl. Exemplary 6-membered heteroarylscontaining 1 heteroatom include, without limitation, pyridinyl.Exemplary 6-membered heteroaryls containing 2 heteroatoms include,without limitation, pyridazinyl, pyrimidinyl and pyrazinyl. Exemplary6-membered heteroaryls containing 3 or 4 heteroatoms include, withoutlimitation, triazinyl and tetrazinyl, respectively. Exemplary 7 memberedheteroaryls containing 1 heteroatom include, without limitation,azepinyl, oxepinyl and thiepinyl. Exemplary 5,6-bicyclic heteroarylsinclude, without limitation, indolyl, isoindolyl, indazolyl,benzotriazolyl, benzothiophenyl, isobenzothiophenyl, benzofuranyl,benzoisofuranyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl,benzoxadiazolyl, benzthiazolyl, benzisothiazolyl, benzthiadiazolyl,indolizinyl, and purinyl. Exemplary 6,6-bicyclic heteroaryls include,without limitation, naphthyridinyl, pteridinyl, quinolinyl,isoquinolinyl, cinnolinyl, quinoxalinyl, phthalazinyl and quinazolinyl.Exemplary tricyclic heteroaryls include, without limitation,phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl,phenoxazinyl and phenazinyl. Unless otherwise specified, each instanceof a heteroaryl group is independently unsubstituted (an “unsubstitutedheteroaryl”) or substituted (a “substituted heteroaryl”) with 1, 2, 3,4, or 5 substituents as described herein. In certain embodiments, theheteroaryl group is an unsubstituted 5-14 membered heteroaryl. Incertain embodiments, the heteroaryl group is a substituted 5-14 memberedheteroaryl.

As used herein, alone or part of another group, “heteroaralkyl” refersto a C₁₋₁₀ alkyl group as defined herein substituted by a 5-14 memberedheteroaryl group as defined herein, wherein the point of attachment ison the alkyl group (“C₁₋₁₀ heteroaralkyl”).

As used herein, a “covalent bond” or “direct bond” refers to a singlebond joining two groups.

As used herein, the term “partially unsaturated” refers to a ring moietythat includes at least one double or triple bond. The term “partiallyunsaturated” is intended to encompass rings having multiple sites ofunsaturation, but is not intended to include aryl or heteroarylmoieties, as herein defined.

As used herein a “divalent” group, such as a divalent alkyl, divalentalkenyl, divalent alkynyl, divalent heteroaliphatic, divalentcarbocyclyl, divalent heterocyclyl, divalent aryl or divalent heteroarylgroup, refers to a bis-radical of the group, as defined herein.

Monovalent or divalent alkyl, alkenyl, alkynyl, heteroaliphatic,carbocyclyl, heterocyclyl, aryl and heteroaryl groups, as definedherein, are either “substituted” or “unsubstituted” alkyl, “substituted”or “unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl,“substituted” or “unsubstituted” heteroaliphatic, “substituted” or“unsubstituted” carbocyclyl, “substituted” or “unsubstituted”heterocyclyl, “substituted” or “unsubstituted” aryl or “substituted” or“unsubstituted” heteroaryl groups. In general, the term “substituted”means that at least one hydrogen present on a group (e.g., a carbon ornitrogen atom, etc.) is replaced with a permissible substituent, e.g., asubstituent which upon substitution results in a stable compound, e.g.,a compound which does not spontaneously undergo transformation such asby rearrangement, cyclization, elimination, or other reaction. Unlessotherwise indicated, a “substituted” group can have a substituent at oneor more substitutable positions of the group, and when more than oneposition in any given structure is substituted, the substituent iseither the same or different at each position. A group referred to as“not hydrogen” indicates that the group is an exemplary and permissiblesubstituent as described herein.

Exemplary substituents include, but are not limited to, halogen (i.e.,fluoro (—F), bromo (—Br), chloro (—Cl), and iodo (—I)), —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂, —N(R^(bb))₂,—N(OR^(cc))R^(bb), —SH, —SR, —SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO,—C(OR^(cc))₂, —CO₂R^(aa), —OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂,—OC(═O)N(R^(bb))₂, —NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa),—NR^(bb)C(═O)N(R^(bb))₂, —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂, —OC(═NR^(bb))N(R^(bb))₂,—NR^(bb)(═NR^(bb))N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa), —NR^(bb)SO₂R^(aa),—SO₂N(R^(bb))₂, —SO₂R^(aa), —SO₂OR^(aa), —OSO₂R^(aa), —S(═O)R^(aa),—OS(═O)R^(aa), —Si(R^(aa))₃, —OSi(R^(aa))₃—C(═S)N(R^(bb))₂,—C(═O)SR^(aa), —C(═S)SR^(aa), —SC(S)SR^(aa), —P(═O)₂R^(aa),—OP(═O)₂R^(aa), —P(═O)(R^(aa))₂, —OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂,—P(═O)₂N(R^(bb))₂, —OP(═O)₂N(R^(bb))₂, —P(═O)(NR^(bb))₂,—OP(═O)(NR^(bb))₂, —NR^(bb)P(═O)(OR^(cc))₂, —NR^(bb)P(═O)(NR^(bb))₂,—P(R^(cc))₂, —P(R^(cc))₃, —OP(R^(cc))₂, —OP(R^(cc))₃, —B(OR^(cc))₂, or—BR(OR^(cc)), ═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(O)R^(aa),═NNR^(bb)CO₂R^(aa), ═NNR^(bb)S(O)₂R^(aa), ═NR^(bb), ═NOR^(cc), C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl,alkynyl, carbocyclyl, heterocyclyl, aryl, and heteroaryl isindependently unsubstituted or substituted with 1-5 R^(dd) groups;

wherein:

each instance of R^(aa) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, wherein each alkyl, alkenyl,alkynyl, heteroaliphatic, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently unsubstituted or substituted with 1-5 R^(dd)groups;

each instance of R^(bb) is, independently, selected from hydrogen, —OH,—OR^(aa), —N(R)₂, —CN, —C(═O)R^(aa), —C(═O)N(R^(cc))₂, —CO₂R^(aa),—SO₂R^(aa), —C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂,—SO₂R^(cc), —SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc),—C(═S)SR^(cc), —P(═O)₂R^(aa), —P(═O)(R^(aa))₂, —P(═O)₂N(R^(cc))₂,—P(═O)(NR)₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(bb)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroaliphatic,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlyunsubstituted or substituted with 1-5 R^(dd) groups;

each instance of R^(cc) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, or two R^(cc) groups are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, heteroaliphatic carbocyclyl,heterocyclyl, aryl, and heteroaryl is independently unsubstituted orsubstituted with 1-5 R^(dd) groups;

each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R)₂, —N(R^(ff))₂,—N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee), —C(O)R^(ee), —CO₂H,—CO₂R^(ee), —OC(O)R^(ee), —OCO₂R^(ee), —C(O)N(R^(ff))₂,—OC(O)N(R^(ff))₂, —NR^(ff)C(O)R^(ee), —NR^(ff)CO₂R^(ee),—NR^(ff)C(O)N(R^(ff))₂, —C(NR^(ff))OR^(ee), —OC(NR^(ff))R^(ee),—OC(NR^(ff))OR^(ee), —C(NR^(ff))N(R^(ff))₂, —OC(NR^(ff))N(R^(ff))₂,—NR^(ff)C(NR^(ff))N(R^(ff))₂, —NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂,—SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee), —SOR^(ee), —Si(R^(ee))₃,—OSi(R^(ee))₃, —C(S)N(R^(ff))₂, —C(O)SR^(ee), —C(S)SR^(ee),—SC(S)SR^(ee), —P(O)₂R^(ee), —P(O)(R^(ee))₂, —OP(O)(R^(ee))₂,—OP(O)(OR^(ee))₂, ═O, ═S, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-10membered heterocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, heteroaliphatic, carbocyclyl,heterocyclyl, aryl, and heteroaryl is independently unsubstituted orsubstituted with 1-5 R^(gg) groups;

each instance of R^(ee) is, independently, selected from C₁₋₆ alkyl,C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl, 3-10 memberedheterocyclyl, and 3-10 membered heteroaryl, wherein each alkyl, alkenyl,alkynyl, heteroaliphatic, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently unsubstituted or substituted with 1-5 R^(gg)groups;

each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-10 membered heterocyclyl, C₆₋₁₀aryl and 5-10 membered heteroaryl, or two R^(ff) groups are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring,wherein each alkyl, alkenyl, alkynyl, heteroaliphatic, carbocyclyl,heterocyclyl, aryl, and heteroaryl is independently unsubstituted orsubstituted with 1-5 R^(gg) groups; and

each instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂,—N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH, —S(C₁₋₆alkyl), —SS(C₁₋₆ alkyl), —C(O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆ alkyl),—OC(O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(O)NH₂, —C(O)N(C₁₋₆ alkyl)₂,—OC(O)NH(C₁₋₆ alkyl), —NHC(O)(C₁₋₆ alkyl), —N(C₁₋₆ alkyl)C(O)(C₁₋₆alkyl), —NHCO₂(C₁₋₆ alkyl), —NHC(O)N(C₁₋₆ alkyl)₂, —NHC(O)NH(C₁₋₆alkyl), —NHC(O)NH₂, —C(NH)O(C₁₋₆ alkyl), —OC(NH)(C₁₋₆ alkyl),—OC(NH)OC₁₋₆ alkyl, —C(NH)N(C₁₋₆ alkyl)₂, —C(NH)NH(C₁ alkyl), —C(NH)NH₂,—OC(NH)N(C₁₋₆ alkyl)₂, —OC(NH)NH(C₁₋₆ alkyl), —OC(NH)NH₂, —NHC(NH)N(C₁₋₆alkyl)₂, —NHC(NH)NH₂, —NHSO₂(C₁₋₆ alkyl), —SO₂N(C₁₋₆ alkyl)₂,—SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂C₁₋₆ alkyl, —SO₂OC₁₋₆ alkyl, —OSO₂C₁₋₆alkyl, —SOC₁₋₆ alkyl, —Si(C₁₋₆ alkyl)₃, —OSi(C₁₋₆ alkyl)₃, —C(S)N(C₁₋₆alkyl)₂, —C(S)NH(C₁₋₆ alkyl), —C(S)NH₂, —C(O)S(C₁₋₆ alkyl), —C(S)SC₁₋₆alkyl, —SC(S)SC₁₋₆ alkyl, —P(O)₂(C₁₋₆ alkyl), —P(O)(C₁₋₆ alkyl)₂,—OP(O)(C₁₋₆ alkyl)₂, —OP(O)(OC₁₋₆ alkyl)₂, C₁₋₆ alkyl, C₁₋₆perhaloalkyl, C₂₋₆ alkenyl, C₂-alkynyl, 3-14 membered heteroaliphatic,C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 memberedheteroaryl, ═O or ═S.

These and other exemplary substituents are described in more detail inthe Detailed Description, the Exemplification and in the claims. Theterm “substituents” is not intended to be limited in any manner by theabove exemplary listing of substituents.

As used herein, a “pharmaceutically acceptable form thereof” includespharmaceutically acceptable salts, hydrates, solvates, prodrugs,tautomers, isomers, and/or polymorphs of a compound provided herein, asdefined below and herein.

In certain embodiments, the pharmaceutically acceptable form thereof isa pharmaceutically acceptable salt. As used herein, the term“pharmaceutically acceptable salt” refers to those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, Berge et al. describespharmaceutically acceptable salts in detail in J. PharmaceuticalSciences (1977) 66:1-19. Pharmaceutically acceptable salts of thecompounds provided herein include those derived from suitable inorganicand organic acids and bases. Examples of pharmaceutically acceptable,nontoxic acid addition salts are salts of an amino group formed withinorganic acids such as hydrochloric acid, hydrobromic acid, phosphoricacid, sulfuric acid and perchloric acid or with organic acids such asacetic acid, oxalic acid, maleic acid, tartaric acid, citric acid,succinic acid or malonic acid or by using other methods used in the artsuch as ion exchange. Other pharmaceutically acceptable salts includeadipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptonate, glycerophosphate, gluconate,hemisulfate, heptanoate, hexanoate, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and thelike. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium and N⁺(C₁₋₄alkyl)₄ salts. Representativealkali or alkaline earth metal salts include sodium, lithium, potassium,calcium, magnesium, and the like. Further pharmaceutically acceptablesalts include, when appropriate, nontoxic ammonium, quaternary ammonium,and amine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate.

In certain embodiments, the pharmaceutically acceptable form thereof isa hydrate or solvate. As used herein, the term “hydrate” refers to acompound non-covalently associated with one or more molecules of water,which in some embodiments can be crystalline. Likewise, “solvate” refersto a compound non-covalently associated with one or more molecules of anorganic solvent, which in some embodiments can be crystalline.

In certain embodiments, the pharmaceutically acceptable form thereof isa prodrug. As used herein, the term “prodrug” refers to a derivative ofa parent compound that requires transformation within the body in orderto release the parent compound.

The term “prodrug” refers to compounds that are transformed in vivo toyield a disclosed compound or a pharmaceutically acceptable form of thecompound. The transformation can occur by various mechanisms, such as,but not limited to, through hydrolysis in blood. In certain cases, aprodrug has improved physical and/or delivery properties over the parentcompound. Prodrugs are typically designed to enhance pharmaceuticallyand/or pharmacokinetically based properties associated with the parentcompound. Exemplary advantages of a prodrug can include, but are notlimited to, its physical properties, such as enhanced water solubilityfor parenteral administration at physiological pH compared to the parentcompound, or it enhances absorption from the digestive tract, or it canenhance drug stability for long-term storage.

For example, if a disclosed compound or a pharmaceutically acceptableform of the compound contains a carboxylic acid functional group, aprodrug can comprise an ester formed by the replacement of the hydrogenatom of the acid group with a group such as (C₁-C₈)alkyl,(C₂-C₁₂)alkanoyloxymethyl, 1-(alkanoyloxy)ethyl having from 4 to 9carbon atoms, 1-methyl-1-(alkanoyloxy)-ethyl having from 5 to 10 carbonatoms, alkoxycarbonyloxymethyl having from 3 to 6 carbon atoms,1-(alkoxycarbonyloxy)ethyl having from 4 to 7 carbon atoms,1-methyl-1-(alkoxycarbonyloxy)ethyl having from 5 to 8 carbon atoms,N-(alkoxycarbonyl)aminomethyl having from 3 to 9 carbon atoms,1-(N-(alkoxycarbonyl)amino)ethyl having from 4 to 10 carbon atoms,3-phthalidyl, 4-crotonolactonyl, gamma-butyrolacton-4-yl,di-N,N—(C₁-C₂)alkylamino(C₂-C₃)alkyl (such as β-dimethylaminoethyl),carbamoyl-(C₁-C₂)alkyl, N,N-di(C₁-C₂)alkylcarbamoyl-(C₁-C₂)alkyl andpiperidino-, pyrrolidino- or morpholino(C₂-C₃)alkyl.

Similarly, if a disclosed compound or a pharmaceutically acceptable formof the compound contains an alcohol functional group, a prodrug can beformed by the replacement of the hydrogen atom of the alcohol group witha group such as (C₁-C₆)alkanoyloxymethyl, 1-((C₁-C₆)alkanoyloxy)ethyl,1-methyl-1-((C₁-C₆)alkanoyloxy)ethyl (C₁-C₆)alkoxycarbonyloxymethyl,N—(C₁-C₆)alkoxycarbonylaminomethyl, succinoyl, (C₁-C₆)alkanoyl,α-amino(C₁-C₄)alkanoyl, arylacyl and α-aminoacyl, orα-aminoacyl-α-aminoacyl, where each α-aminoacyl group is independentlyselected from the naturally occurring L-amino acids, P(O)(OH)₂,—P(O)(O(C₁-C₆)alkyl)₂ or glycosyl (the radical resulting from theremoval of a hydroxyl group of the hemiacetal form of a carbohydrate).

If a disclosed compound or a pharmaceutically acceptable form of thecompound incorporates an amine functional group, a prodrug can be formedby the replacement of a hydrogen atom in the amine group with a groupsuch as R-carbonyl, RO-carbonyl, NRR′-carbonyl where R and R′ are eachindependently (C₁-C₁₀)alkyl, (C₃-C₇)cycloalkyl, benzyl, or R-carbonyl isa natural α-aminoacyl or natural α-aminoacyl-natural α-aminoacyl,C(OH)C(O)OY¹ wherein Y¹ is H, (C₁-C₆)alkyl or benzyl, —C(OY²)Y³ whereinY² is (C₁-C₄) alkyl and Y³ is (C₁-C₆)alkyl, carboxy(C₁-C₆)alkyl,amino(C₁-C₄)alkyl or mono-N— or di-N,N—(C₁-C₆)alkylaminoalkyl, —C(Y⁴)Y⁵wherein Y⁴ is H or methyl and Y⁵ is mono-N— or di-N,N—(C₁-C₆)alkylamino,morpholino, piperidin-1-yl or pyrrolidin-1-yl.

In certain embodiments, the pharmaceutically acceptable form thereof isa tautomer. As used herein, the term “tautomer” includes two or moreinterconvertable compounds resulting from at least one formal migrationof a hydrogen atom and at least one change in valency (e.g., a singlebond to a double bond, a triple bond to a single bond, or vice versa).The exact ratio of the tautomers depends on several factors, includingtemperature, solvent, and pH. Tautomerizations (i.e., the reactionproviding a tautomeric pair) can be catalyzed by acid or base, or canoccur without the action or presence of an external agent. Exemplarytautomerizations include, but are not limited to, keto-to-enol;amide-to-imide; lactam-to-lactim; enamine-to-imine; and enamine-to-(adifferent) enamine tautomerizations.

In certain embodiments, the pharmaceutically acceptable form thereof isan isomer. As used herein, the term “isomer” includes any and allgeometric isomers and stereoisomers. For example, “isomers” include cis-and trans-isomers, E- and Z-isomers, R- and S-enantiomers,diastereomers, (D)-isomers, (L)-isomers, racemic mixtures thereof, andother mixtures thereof, as falling within the scope of this disclosure.For instance, an isomer/enantiomer can, in some embodiments, be providedsubstantially free of the corresponding enantiomer, and may also bereferred to as “optically enriched.” “Optically-enriched,” as usedherein, means that the compound is made up of a significantly greaterproportion of one enantiomer. In certain embodiments, the compoundprovided herein is made up of at least about 90% by weight of oneenantiomer. In other embodiments the compound is made up of at leastabout 95%, 98%, or 99% by weight of one enantiomer. Enantiomers can beisolated from racemic mixtures by any method known to those skilled inthe art, including chiral high pressure liquid chromatography (HPLC),the formation and crystallization of chiral salts, or prepared byasymmetric syntheses. See, for example, Enantiomers, Racemates andResolutions (Jacques, Ed., Wiley Interscience, New York, 1981); Wilen etal., Tetrahedron 33:2725 (1977); Stereochemistry of Carbon Compounds (E.L. Eliel, Ed., McGraw-Hill, N Y, 1962); and Tables of Resolving Agentsand Optical Resolutions p. 268 (E. L. Eliel, Ed., Univ. of Notre DamePress, Notre Dame, Ind. 1972).

In certain embodiments, the pharmaceutically acceptable form thereof isa polymorph. As used herein, “polymorph” refers to a compound havingmore than one crystal structure, e.g., resulting from differences inmolecular packing and/or molecular conformation of the compound in thesolid state.

The disclosure also embraces isotopically labeled compounds which areidentical to those recited herein, except that one or more atoms arereplaced by an atom having an atomic mass or mass number different fromthe atomic mass or mass number usually found in nature. Examples ofisotopes that can be incorporated into disclosed compounds includeisotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, fluorine andchlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ¹⁷O, ³¹P, ³²P, ³⁵S, ¹⁸F,and ³⁶Cl, respectively.

Certain isotopically-labeled disclosed compounds (e.g., those labeledwith ³H and ¹⁴C) are useful in compound and/or substrate tissuedistribution assays. Tritiated (i.e., ³H) and carbon-14 (i.e., ¹⁴C)isotopes acan allow for ease of preparation and detectability. Further,substitution with heavier isotopes such as deuterium (i.e., ²H) canafford certain therapeutic advantages resulting from greater metabolicstability (e.g., increased in vivo half-life or reduced dosagerequirements). Isotopically labeled disclosed compounds can generally beprepared by following procedures analogous to those disclosed in theExemplification section herein by substituting an isotopically labeledreagent for a non-isotopically labeled reagent.

DETAILED DESCRIPTION 1. Brief Description of Figures

FIG. 1 illustrates a schematic diagram of subcutaneous mouse xenograftmodel for assaying papillomaviruses.

FIG. 2 illustrates a schematic diagram of cutaneous mouse xenograftmodel for assaying papillomaviruses.

2. Compounds

Without being limited by a particular theory, the present disclosure isbased on the discovery that tetrazolones are inhibitors of human fattyacid synthase (FASN) and thus are useful in the treatment ofFASN-mediated diseases, disorders or conditions. Further, without beinglimited by a particular theory, in certain embodiments, the compoundsprovided herein can inhibit long chain fatty acid elongase (ELOVL) suchas ELOVL 6. Thus, in some embodiments, compounds provided herein areuseful in the treatment of ELOVL-mediated diseases, disorders orconditions.

For example, in one aspect, provided herein is a compound of formula(I):

or a pharmaceutically acceptable form thereof;

wherein:

R^(A) is selected from C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl and 5-14 membered heteroaryl;

R^(B) is selected from C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl and 5-14 membered heteroaryl;

R^(C) is selected from hydrogen, —OH, —OR^(C1), —ON(R^(C2))₂,—N(R^(C2))₂, —C(═O)R^(C1), —CHO, —CO₂R^(C1), —C(═O)N(R^(C2))₂,—C(═NR^(C2))OR^(C1), —C(═NR^(C2))N(R^(C2))₂, —SO₂R^(C1), —S(═O)R^(C1),—Si(R^(C1))₃, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl;

wherein:

each instance of R^(C1) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl; and each instance of R^(C2) is,independently, selected from hydrogen, —OH, —OR^(C1), —N(R^(C3))₂, —CN,—C(═O)R^(C1), —C(═O)N(R^(C3))₂, —CO₂R^(C1), —SO₂R^(C1),—C(═NR^(C3))OR^(C1), —C(═NR^(C3))N(R^(C3))₂, —SO₂N(R^(C3))₂, —SO₂R^(C3),—SO₂OR^(C3), —SOR^(C1), —C(═S)N(R^(C3))₂, —C(═O)SR^(C3), —C(═S)SR^(C3),—P(═O)₂R^(C1), —P(═O)(R^(C1))₂, —P(═O)₂N(R^(C3))₂, —P(═O)(NR^(C3))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(C2) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring;

or R^(B) and R^(C) together with the nitrogen (N) atom to which each isattached are joined to form a 5-14 membered heterocyclyl or heteroarylring.

In one embodiment, provided herein is a compound of formula (I):

or a pharmaceutically acceptable form thereof;

wherein:

R^(A) is selected from C₆₋₁₄ aryl and 5-14 membered heteroaryl;

R^(B) is selected from C₆₋₁₄ aryl and 5-14 membered heteroaryl;

R^(C) is selected from —OH, —OR^(C1), —ON(R^(C2))₂, —N(R^(C2))₂,—C(═O)R^(C1), —CHO, —CO₂R^(C1), —C(═O)N(R^(C2))₂, —C(═NR^(C2))OR^(C1),—C(═NR^(C2))N(R^(C2))₂, —SO₂R^(C1), —S(═O)R^(C1), —Si(R^(C1))₃, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, with the proviso that R^(C) is not—CH₃;

each instance of R^(C1) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl;

each instance of R^(C2) is, independently, selected from hydrogen, —OH,—OR^(C1), —N(R^(C3))₂, —CN, —C(═O)R^(C1), —C(═O)N(R^(C3))₂, —CO₂R^(C1),—SO₂R^(C1), —C(═NR^(C3))OR^(C1), —C(═NR^(C3))N(R^(C3))₂, —SO₂N(R^(C3))₂,—SO₂R^(C3), —SO₂OR^(C3), —SOR^(C1), —C(═S)N(R^(C3))₂, —C(═O)SR^(C3),—C(═S)SR^(C3), —P(═O)₂R^(C1), —P(═O)(R^(C1))₂, —P(═O)₂N(R^(C3))₂,—P(═O)(NR^(C3))₂, C₂₋₁₀ alkyl, C₂₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(C2)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring;

or R^(B) and R^(C) together with the nitrogen (N) atom to which each isattached are joined to form a 5-14 membered ring;

wherein:

R^(B) is substituted with the group:

-L-R^(D)

wherein:

L is a covalent bond or a divalent C₁₋₁₀ hydrocarbon chain, wherein one,two or three methylene units of L are optionally and independentlyreplaced with one or more —O—, —S—, —NR^(B8)—, —(C═NR^(B8))—, —C(═O)—,—C(═S)—, —S(═O)—, —S(═O)₂—, divalent carbocyclyl, divalent heterocyclyl,divalent aryl or divalent heteroaryl group;

R^(D) is selected from —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —C(═O)R^(B7),—CO₂H, —CHO, —C(OR^(B9))₂, —CO₂R^(B7), —OC(═O)R^(B7), —OCO₂R^(B7),—C(═O)N(R^(B8))₂, —OC(═O)N(R^(B8))₂, —NR^(B8)C(═O)R^(B7),—NR^(B8)CO₂R^(B7), —NR^(B8)C(═O)N(R^(B8))₂, —C(═NR^(B8))OR^(B7),—OC(═NR^(B8))R^(B7), —OC(═NR^(B8))OR^(B7), —C(═NR^(B8))N(R^(B8))₂,—OC(═NR^(B8))N(R^(B8))₂, —NR^(B8)C(═NR^(B8))N(R^(B8))₂,—C(═O)NR^(B8)SO₂R, —NR^(B8)SO₂R^(B7), —SO₂N(R^(B8))₂, —SO₂R^(B7),—SO₂OR^(B7), —OSO₂R^(B7), —S(═O)R^(B7), —OS(═O)R^(B7), —C(═S)N(R^(B8))₂,—C(═O)SR^(B7), —C(═S)SR^(B7), —SC(═S)SR^(B7), —P(═O)₂R^(B7),—OP(═O)₂R^(B7), —P(═O)(R^(B7))₂, —OP(═O)(R^(B7))₂, —OP(═O)(OR^(B9))₂,—P(═O)₂N(R^(B8))₂, —OP(═O)₂N(R^(B8))₂, —P(═O)(NR^(B8))₂,—OP(═O)(NR^(B8))₂, —NR^(B8)P(═O)(OR^(B9))₂, —NR^(B8)P(═O)(NR^(B8))₂,—B(OR^(B9))₂, —BR^(B7)(OR^(B9)), and tetrazolyl;

each instance of R^(B7) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl;

each instance of R^(B8) is, independently, selected from hydrogen, —OH,—OR^(B7), —N(R^(B9))₂, —CN, —C(═O)R^(B7), —C(═O)N(R^(B9))₂, —CO₂R^(B7),—SO₂R^(B7), —C(═NR^(B9))OR^(B7), —C(═NR^(B9))N(R^(B9))₂, —SO₂N(R^(B9))₂,—SO₂R^(B9), —SO₂OR^(B9), —SOR^(B7), —C(═S)N(R^(B9))₂, —C(═O)SR^(B9),—C(═S)SR^(B9), —P(═O)₂R^(B7), —P(═O)(R^(B7))₂, —P(═O)₂N(R^(B9))₂,—P(═O)(NR^(B9))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B8)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring; and

each instance of R^(B9) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, or two R^(B9) groups are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring.

In one embodiment, provided herein is a compound of formula (I):

or a pharmaceutically acceptable form thereof;

wherein:

R^(A) is selected from C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl and 5-14 membered heteroaryl;

R^(B) is selected from C₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl and 5-14 membered heteroaryl;

R^(C) is selected from hydrogen, —OH, —OR^(C1), —ON(R^(C2))₂,—N(R^(C2))₂, —C(═O)R^(C1), —CHO, —CO₂R^(C1), —C(═O)N(R^(C2))₂,—C(═NR^(C2))OR^(C1), —C(═NR^(C2))N(R^(C2))₂, —SO₂R^(C1), —S(═O)R^(C1),—Si(R^(C1))₃, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl;

each instance of R^(C1) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl; and

each instance of R^(C2) is, independently, selected from hydrogen, —OH,—OR^(C1), —N(R^(C3))₂, —CN, —C(═O)R^(C1), —C(═O)N(R^(C3))₂, —CO₂R^(C1),—SO₂R^(C1), —C(═NR^(C3))OR^(C1), —C(═NR^(C3))N(R^(C3))₂, —SO₂N(R^(C3))₂,—SO₂R^(C3), —SO₂OR^(C3), —SOR^(C1), —C(═S)N(R^(C3))₂, —C(═O)SR^(C3),—C(═S)SR^(C3), —P(═O)₂R^(C1), —P(═O)(R^(C1))₂, —P(═O)₂N(R^(C3))₂,—P(═O)(NR^(C3))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(C2)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring;

or R^(B) and R^(C) together with the nitrogen (N) atom to which each isattached are joined to form a 5-14 membered ring.

Group R^(A)

As described generally above, R^(A) is selected from C₃₋₁₀ carbocyclyl,3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl.

In certain embodiments, R^(A) is C₃₋₁₀ carbocyclyl. Exemplarycarbocyclyl groups include, but are not limited to, cyclopropyl (C₃),cyclobutyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅), cyclohexyl (C₆),cyclohexenyl (C₆), cyclohexadienyl (C₆), cycloheptyl (C₇),cycloheptadienyl (C₇), cycloheptatrienyl (C₇) and cyclooctyl (C₈).

In certain embodiments, R^(A) is 3-14 membered heterocyclyl. Exemplaryheterocyclyl groups include, but are not limited to, azirdinyl,oxiranyl, thiorenyl, azetidinyl, oxetanyl, thietanyl, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl,dihydropyrrolyl, dioxolanyl, oxathiolanyl, dithiolanyl, piperidinyl,tetrahydropyranyl, dihydropyridinyl, thianyl, piperazinyl, morpholinyl,dithianyl, dioxanyl, azepanyl, oxepanyl thiepanyl, azocanyl, oxecanyland thiocanyl.

In certain embodiments, R^(A) is C₆₋₁₄ aryl. Exemplary aryl groupsinclude, but are not limited to, phenyl, naphthyl and anthracyl. Incertain embodiments, R^(A) is phenyl (C₆ aryl). In certain embodiments,R^(A) is naphthyl (C₁₀ aryl).

In certain embodiments, R^(A) is 5-14 membered heteroaryl. In certainembodiments, R^(A) is 5-10 membered heteroaryl. In certain embodiments,R^(A) is 5-6 membered heteroaryl. In certain embodiments, R^(A) is5,6-bicyclic heteroaryl. In certain embodiments, R^(A) is 6,6-bicyclicheteroaryl.

In certain embodiments, R^(A) is a 5-membered heteroaryl group.Exemplary 5-membered heteroaryl groups include, but are not limited to,pyrrolyl, furanyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl,thiadiazolyl and tetrazolyl.

In certain embodiments, R^(A) is a 6-membered heteroaryl group.Exemplary 6-membered heteroaryl groups include, but are not limited to,pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl andtetrazinyl.

In certain embodiments, R^(A) is a 5,6-bicyclic heteroaryl group.Exemplary 5,6-bicyclic heteroaryl groups include, but are not limitedto, indolyl, isoindolyl, indazolyl, benztriazolyl, benzothiophenyl,isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl,benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl,benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.

In certain embodiments, R^(A) is a 6,6-bicyclic heteroaryl group.Exemplary 6,6-bicyclic heteroaryl groups include, but are not limitedto, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl,quinoxalinyl, phthalazinyl and quinazolinyl.

In certain embodiments, R^(A) is a group of the formula (i):

wherein each group W—R¹, W—R², W—R³, W—R⁴, and W—R⁵ independentlyrepresents either a nitrogen atom (N) or C—R¹, C—R², C—R³, C—R⁴, orC—R⁵, respectively; and

wherein R¹, R², R³, R⁴ and R⁵ are independently selected from the groupconsisting of hydrogen, halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH,—OR^(A1), —ON(R^(A2))₂, —N(R^(A2))₂, —N(OR^(A3))R^(A3), —SH, —SR^(A1),—SSR^(A3), —C(═O)R^(A1), —CO₂H, —CHO, —C(OR^(A3))₂, —CO₂R^(A1),—OC(═O)R^(A1), —OCO₂R^(A1), —C(═O)N(R^(A2))₂, —OC(═O)N(R^(A2))₂,—NR^(A2)C(═O)R^(A1), —NR^(A2)C₂R^(A1), —NR^(A2)C(═O)N(R^(A2))₂,—C(═NR^(A2))OR^(A1), —OC(═NR)R^(A1), —OC(═NR^(A2))OR^(A1),—C(═NR^(A2))N(R^(A2))₂, —OC(═NR^(A2))N(R^(A2))₂,—NR^(A2)C(═NR^(A2))N(R^(A2))₂, —C(═O)NR^(A2)SO₂R^(A1),—NR^(A2)SO₂R^(A1), —SO₂N(R^(A2))₂, —SO₂R^(A1), —SO₂OR^(A1), —OSO₂R^(A1),—S(═O)R^(A1), —OS(═O)R^(A1), Si(R^(A1))₃, —OSi(R^(A1))₃—C(═S)N(R^(A2))₂,—C(═O)SR^(A1), —C(═S)SR^(A1), —SC(═S)SR^(A1), —P(═O)₂R^(A1),—OP(═O)₂R^(A1), —P(═O)(R^(A1))₂, —OP(═O)(R^(A1))₂, —OP(═O)(OR^(A3))₂,—P(═O)₂N(R^(A2))₂, —OP(═O)₂N(R^(A2))₂, —P(═O)(NR^(A2))₂,—OP(═O)(NR^(A2))₂, —NR^(A2)P(═O)(OR^(A3))₂, —NR^(A2)P(═O)(NR^(A2))₂,—P(R^(A3))₂, —P(R^(A3))₃, —OP(R^(A3))₂, —OP(R^(A3))₃, —B(OR^(A3))₂,—BR^(A1)(OR^(A3)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl; or one or moreof R¹ and R², R² and R³, R³ and R⁴ or R⁴ and R⁵ are joined to form aC₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl or 5-14membered heteroaryl ring;

each instance of R^(A)1 is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl;

each instance of R^(A2) is, independently, selected from hydrogen, —OH,—OR^(A1), —N(R^(A3))₂, —CN, —C(═O)R^(A1), —C(═O)N(R^(A3))₂, —CO₂R^(A1),—SO₂R^(A1), —C(═NR^(A3))OR^(A1), —C(═NR^(A3))N(R^(A3))₂, —SO₂N(R^(A3))₂,—SO₂R^(A3), —SO₂OR^(A3), —SOR^(A1), —C(═S)N(R^(A3))₂, —C(═O)SR^(A3),—C(═S)SR^(A3), —P(═O)₂R^(A1), —P(═O)(R^(A1))₂, —P(═O)₂N(R^(A3))₂,—P(═O)(NR^(A3))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(A2)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring; and

each instance of R^(A3) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, or two R^(A3) groups are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring.

In certain embodiments, the group of formula (i) represents a C₆₋₁₄ arylgroup or a 6-14 membered heteroaryl group. In certain embodiments, thegroup of formula (i) represents a 6-14 membered heteroaryl group. Incertain embodiments, the group of formula (i) represents a C₆₋₁₄ arylgroup. In certain embodiments, the C₆₋₁₄ aryl group of formula (i)represents a phenyl group.

As used herein, when one or more of R¹, R², R³, R⁴ and R⁵ is referred toas “not hydrogen”, it is meant that one or more of R¹, R², R³, R⁴ and R⁵is independently selected from a group consisting of halogen, —CN, —NO₂,—N₃, —SO₂H, —SO₃H, —OH, —OR^(A1), —ON(R^(A2))₂, —N(R^(A2))₂,—N(OR^(A3))R^(A3), —SH, —SR^(A1), —SSR^(A3), —C(═O)R^(A1), —CO₂H, —CHO,—C(OR^(A3))₂, —CO₂R^(A1), —OC(═O)R^(A1), —OCO₂R^(A1), —C(═O)N(R^(A2))₂,—OC(═O)N(R^(A2))₂, —NR^(A2)C(═O)R^(A1), —NR^(A2)CO₂R^(A1),—NR^(A2)C(═O)N(R^(A2))₂, —C(═NR^(A2))OR^(A1), —OC(═NR^(A2))R^(A1),—OC(═NR^(A2))OR^(A1), —C(═NR^(A2))N(R^(A2))₂, —OC(═NR^(A2))N(R^(A2))₂,—NR^(A2)C(═NR^(A2))N(R^(A2))₂, —C(═O)NR^(A2)SO₂R^(A1),—NR^(A2)SO₂R^(A1), —SO₂N(R^(A2))₂, —SO₂R^(A1), —SO₂OR^(A1), —OSO₂R^(A1),—S(═O)R^(A1), —OS(═O)R^(A1), Si(R^(A1))₃, —OSi(R^(A1))₃—C(═S)N(R^(A2))₂,—C(═O)SR^(A1), —C(═S)SR^(A1), —SC(S)SR^(A1), —P(═O)₂R^(A1),—OP(═O)₂R^(A1), —P(═O)(R^(A1))₂, —OP(═O)(R^(A1))₂, —OP(═O)(OR^(A3))₂,—P(═O)₂N(R^(A2))₂, —OP(═O)₂N(R^(A2))₂, —P(═O)(NR^(A2))₂,—OP(═O)(NR^(A2))₂, —NR^(A2)P(═O)(OR^(A3))₂, —NR^(A2)P(═O)(NR^(A2))₂,—P(R^(A)3)₂, —P(R^(A3))₃, —OP(R^(A3))₂, —OP(R^(A3))₃, —B(OR^(A3))₂, or—BR^(A)(OR^(A3)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl; or one or moreof R¹ and R², R² and R³, R³ and R⁴ or R⁴ and R⁵ are joined to form aC₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl or 5-14membered heteroaryl ring.

In certain embodiments, R¹, R², R³, R⁴ and R⁵ are independently selectedfrom the group consisting of hydrogen, halogen, —CN, —NO₂, —SO₂H, —SO₃H,—OH, —OR^(A1), —N(R^(A2))₂, —C(═O)R^(A1), —CO₂H, —CHO, —C(OR^(A3))₂,—CO₂R^(A1), —OC(═O)R^(A1), —OCO₂R^(A1), —C(═O)N(R^(A2))₂,—OC(═O)N(R^(A2))₂, —NR^(A2)C(═O)R^(A1), —NR^(A2)CO₂R^(A1),—NR^(A2)C(═O)N(R^(A2))₂, —C(═NR^(A2))OR^(A1), —OC(═NR^(A2))R^(A1),—OC(═NR^(A2))OR^(A1), —C(═NR^(A2))N(R^(A2))₂, —OC(═NR^(A2))N(R^(A2))₂,—NR^(A2)C(═NR^(A2))N(R^(A2))₂, —C(═O)NR^(A2)SO₂R^(A1),—NR^(A2)SO₂R^(A1), —SO₂N(R^(A2))₂, —SO₂R^(A1), —SO₂OR^(A1), —OSO₂R^(A1),—S(═O)R^(A1), —OS(═O)R^(A1), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl; or one or more of R¹ and R², R² and R³, R³ and R⁴ or R⁴ andR⁵ are joined to form a C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl or 5-14 membered heteroaryl ring.

In certain embodiments, R¹, R², R³, R⁴ and R⁵ are independently selectedfrom the group consisting of hydrogen, halogen, —CN, —OR^(A1),—N(R^(A2))₂, —CO₂H, —CO₂R^(A1), —C(═O)N(R^(A2))₂, —SO₂R^(A1), C₁₋₁₀alkyl, C₂₋₁₀ alkynyl, 3-14 membered heterocyclyl, and C₆₋₁₄ aryl; or oneor more of R¹ and R², R² and R³, R³ and R⁴ or R⁴ and R⁵ are joined toform a 5-14 membered heteroaryl ring.

In certain embodiments, R¹, R², R³, R⁴ and R⁵ are independently selectedfrom the group consisting of hydrogen, halogen, —OR^(A1), —N(R^(A2))₂,—CO₂H, —C(═O)N(R^(A2))₂, —SO₂R^(A1), and 3-14 membered heterocyclyl; orR⁴ and R⁵ are joined to form a 5-14 membered heteroaryl ring.

In certain embodiments, R¹, R², R³, R⁴ and R⁵ are independently selectedfrom the group consisting of hydrogen, halogen, —OR^(A1), and—C(═O)N(R^(A2))₂; or R⁴ and R⁵ are joined to form a 5-14 memberedheteroaryl ring.

In certain embodiments, R¹, R², R³, R⁴ and R⁵ are independently selectedfrom the group consisting of hydrogen, halogen, —OR^(A1), and—C(═O)N(R^(A2))₂; or R⁴ and R⁵ are joined to form a 5-14 memberedheteroaryl ring.

In certain embodiments, R¹, R², R³, R⁴ and R⁵ are independently selectedfrom the group consisting of hydrogen, halogen, and —OR^(A1). In certainembodiments, R¹, R², R³, R⁴ and R⁵ are independently selected from thegroup consisting of hydrogen, fluoro, chloro, and —OR^(A1). In certainembodiments, R¹, R², R³, R⁴ and R⁵ are independently selected from thegroup consisting of hydrogen, fluoro, chloro, and —OMe. In certainembodiments, R¹, R², R³, R⁴ and R⁵ are independently selected from thegroup consisting of hydrogen, fluoro and —OR^(A1). In certainembodiments, R¹, R², R³, R⁴ and R⁵ are independently selected from thegroup consisting of hydrogen, fluoro and —OMe. In certain embodiments,R¹, R², R³, R⁴ and R⁵ are independently selected from the groupconsisting of hydrogen and fluoro. In certain embodiments, R¹, R², R³,R⁴ and R⁵ are independently selected from the group consisting ofhydrogen and chloro.

In certain embodiments, R⁴ and R⁵ are joined to form a 5-14 memberedheteroaryl ring.

In certain embodiments, R^(A) is a group of the formula (ii):

wherein R¹, R², R³, R⁴ and R⁵ are as defined above and herein.

In certain embodiments, the group of formula (ii) represents a C₆₋₁₄aryl group. In certain embodiments, the C₆₋₁₄ aryl group of formula (ii)represents a phenyl group.

In certain embodiments, R^(A) is a monosubstituted, disubstituted ortrisubstituted group of the formula (ii). In certain embodiments, R^(A)is a monosubstituted or disubstituted group of the formula (ii).

In certain embodiments, R^(A) is a monosubstituted group of the formula(ii).

For example, in certain embodiments, R^(A) is an ortho-substituted groupof the formula (ii), e.g., wherein R¹-R⁴ are hydrogen, and R⁵ is nothydrogen, e.g., of the formula (ii-a).

In certain embodiments, R^(A) is a meta-substituted group of the formula(ii), e.g., wherein R¹-R³ and R⁵ are hydrogen and R⁴ is not hydrogen,e.g., of the formula (ii-b).

In certain embodiments, R^(A) is a para-substituted group of the formula(ii), e.g., wherein R¹, R², R⁴ and R⁵ are hydrogen and R³ is nothydrogen, e.g., of the formula (ii-c).

In certain embodiments, R^(A) is a disubstituted group of the formula(ii).

For example, in certain embodiments, R^(A) is a 2,6-disubstituted groupof the formula (ii), e.g., wherein R², R³ and R⁴ are hydrogen, and R¹and R⁵ are not hydrogen, e.g., of the formula (ii-d).

In certain embodiments, R^(A) is a 2,5-disubstituted group of theformula (ii), e.g., wherein R², R³ and R⁵ are hydrogen, and R¹ and R⁴are not hydrogen, e.g., of the formula (ii-e).

In certain embodiments, R^(A) is a 2,4-disubstituted group of theformula (ii), e.g., wherein R², R³ and R⁵ are hydrogen, and R¹ and R³are not hydrogen, e.g., of the formula (ii-f).

In certain embodiments, R^(A) is a 2,3-disubstituted group of theformula (ii), e.g., wherein R¹, R² and R³ are hydrogen, and R⁴ and R⁵are not hydrogen, e.g., of the formula (ii-g).

In certain embodiments, R^(A) is a 3,4-disubstituted group of theformula (ii), e.g., wherein R¹, R⁴ and R⁵ are hydrogen, and R² and R³are not hydrogen, e.g., of the formula (ii-h).

In certain embodiments, R^(A) is a 3,5-disubstituted group of theformula (ii), e.g., wherein R¹, R³ and R⁵ are hydrogen, and R² and R⁴are not hydrogen, e.g., of the formula (ii-i).

For example, in certain embodiments, R^(A) is a 2,6-disubstituted groupas described herein, e.g., of the formula (ii-d):

wherein R¹ and R⁵ are as defined above and herein.

In certain embodiments, one of R¹ and R⁵ is halogen, —CN, —OR^(A1),—N(R^(A2))₂, —CO₂H, —CO₂R^(A1), —C(═O)N(R^(A2))₂, —SO₂R^(A1), C₁₋₁₀alkyl, C₂₋₁₀ alkynyl, 3-14 membered heterocyclyl, and C₆₋₁₄ aryl, andthe other of R¹ and R⁵ is halogen, —CN, —OR^(A1), —N(R^(A2))₂, —CO₂H,—CO₂R^(A1), —C(═O)N(R^(A2))₂, —SO₂R^(A1), C₁₋₁₀ alkyl, C₂₋₁₀ alkynyl,3-14 membered heterocyclyl, and C₆₋₁₄ aryl.

In certain embodiments, one of R¹ and R⁵ is halogen, —OR^(A1), C₁₋₁₀alkyl, or —C(═O)N(R^(A2))₂, and the other of R¹ and R⁵ is halogen,—OR^(A1), C₁₋₁₀ alkyl, or —C(═O)N(R^(A2))₂.

In certain embodiments, each of R¹ and R⁵ is independently halogen. Forexample, each of R¹ and R⁵ is independently selected from fluoro andchloro.

In certain embodiments, R^(A) is a trisubstituted group of the formula(ii).

For example, in certain embodiments, R^(A) is a 2,4,6-trisubstitutedgroup of the formula (ii), e.g., wherein R² and R⁴ are hydrogen, and R¹,R³ and R⁵ are not hydrogen, e.g., of the formula (ii-j).

In certain embodiments, R^(A) is a 2,3,6-trisubstituted group of theformula (ii), e.g., wherein R² and R³ are hydrogen, and R¹, R⁴ and R⁵are not hydrogen, e.g., of the formula (ii-k).

In certain embodiments, R^(A) is a 2,4,5-trisubstituted group of theformula (ii), e.g., wherein R² and R⁵ are hydrogen, and R¹, R³ and R⁴are not hydrogen, e.g., of the formula (ii-1).

In certain embodiments, R^(A) is a 2,3,4-trisubstituted group of theformula (ii), e.g., wherein R⁴ and R⁵ are hydrogen, and R¹, R² and R³are not hydrogen, e.g., of the formula (ii-m).

In certain embodiments, R^(A) is a 3,4,5-trisubstituted group of theformula (ii), e.g., wherein R¹ and R⁵ are hydrogen, and R², R³ and R⁴are not hydrogen, e.g., of the formula (ii-n).

In certain embodiments, R^(A) is heteroaryl selected from a 5-6-memberedheteroaryl, a 5,6-bicyclic heteroaryl or a 6,6-bicyclic heteroaryl.

In certain embodiments, R^(A) is a 6-membered heteroaryl. In certainembodiments, R^(A) is a 6-membered heteroaryl selected from pyridinyl.In certain embodiments, R^(A) is 2-pyridinyl, 3-pyridinyl or4-pyridinyl.

In certain embodiments, R^(A) is a 2-pyridinyl wherein W—R¹ is N, andW—R², W—R³, W—R⁴, and W—R⁵ are C—R², C—R³, C—R⁴ and C—R⁵, respectively,e.g., of the formula (iii).

In certain embodiments, R^(A) is a 3-pyridinyl wherein W—R² is N, andW—R¹, W—R³, W—R⁴, and W—R⁵ are C—R¹, C—R³, C—R⁴ and C—R⁵, respectively,e.g., of the formula (iv).

In certain embodiments, R^(A) is a 4-pyridinyl wherein W—R³ is N, andW—R¹, W—R², W—R⁴, and W—R⁵ are C—R¹, C—R², C—R⁴ and C—R⁵, respectively,e.g., of the formula (v).

wherein R¹, R², R³, R⁴ and R⁵ are as defined above and herein.

In certain embodiments, R^(A) is a monosubstituted or disubstitutedpyridinyl.

In certain embodiments, R^(A) is a monosubstituted pyridinyl.

In certain embodiments, R^(A) is a monosubstituted pyridinyl of theformula (iii) wherein R³, R⁴, R⁵ are hydrogen and R² is not hydrogen,e.g., of the formula (iii-a).

In certain embodiments, R^(A) is a monosubstituted pyridinyl of theformula (iii) wherein R², R⁴, R⁵ are hydrogen and R³ is not hydrogen,e.g., of the formula (iii-b).

In certain embodiments, R^(A) is a monosubstituted pyridinyl of theformula (iii) wherein R², R³, R⁵ are hydrogen and R⁴ is not hydrogen,e.g., of the formula (iii-c).

In certain embodiments, R^(A) is a monosubstituted pyridinyl of theformula (iii) wherein R², R³, R⁴ are hydrogen and R⁵ is not hydrogen,e.g., of the formula (iii-d).

In certain embodiments, R^(A) is a monosubstituted pyridinyl of theformula (iv) wherein R³, R⁴, R⁵ are hydrogen and R¹ is not hydrogen,e.g., of the formula (iv-a).

In certain embodiments, R^(A) is a monosubstituted pyridinyl of theformula (iv) wherein R¹, R⁴, R⁵ are hydrogen and R³ is not hydrogen,e.g., of the formula (iv-b).

In certain embodiments, R^(A) is a monosubstituted pyridinyl of theformula (iv) wherein R¹, R³, R⁵ are hydrogen and R⁴ is not hydrogen,e.g., of the formula (iv-c).

In certain embodiments, R^(A) is a monosubstituted pyridinyl of theformula (iv) wherein R¹, R³, R⁴ are hydrogen and R⁵ is not hydrogen,e.g., of the formula (iv-d).

In certain embodiments, R^(A) is a monosubstituted pyridinyl of theformula (v) wherein R², R⁴, R⁵ are hydrogen and R¹ is not hydrogen,e.g., of the formula (v-a).

In certain embodiments, R^(A) is a monosubstituted pyridinyl of theformula (v) wherein R¹, R⁴, R⁵ are hydrogen and R² is not hydrogen,e.g., of the formula (v-b).

In certain embodiments, R^(A) is a disubstituted pyridinyl.

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (iii) wherein R³ and R⁴ are hydrogen and R² and R⁵ are nothydrogen, e.g., of the formula (iii-e).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (iii) wherein R² and R⁴ are hydrogen and R³ and R⁵ are nothydrogen, e.g., of the formula (iii-f).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (iii) wherein R² and R³ are hydrogen and R⁴ and R⁵ are nothydrogen, e.g., of the formula (iii-g).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (iii) wherein R³ and R⁵ are hydrogen and R² and R⁴ are nothydrogen, e.g., of the formula (iii-h).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (iii) wherein R⁴ and R⁵ are hydrogen and R² and R³ are nothydrogen, e.g., of the formula (iii-i).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (iii) wherein R² and R⁵ are hydrogen and R³ and R⁴ are nothydrogen, e.g., of the formula (iii-j).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (iv) wherein R³ and R⁴ are hydrogen and R¹ and R⁵ are nothydrogen, e.g., of the formula (iv-e).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (iv) wherein R³ and R⁵ are hydrogen and R¹ and R⁴ are nothydrogen, e.g., of the formula (iv-f).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (iv) wherein R⁴ and R⁵ are hydrogen and R¹ and R³ are nothydrogen, e.g., of the formula (iv-g).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (iv) wherein R¹ and R⁴ are hydrogen and R³ and R⁵ are nothydrogen, e.g., of the formula (iv-h).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (iv) wherein R¹ and R⁵ are hydrogen and R³ and R⁴ are nothydrogen, e.g., of the formula (iv-i).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (iv) wherein R¹ and R³ are hydrogen and R⁴ and R⁵ are nothydrogen, e.g., of the formula (iv-j).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (v) wherein R² and R⁴ are hydrogen and R¹ and R⁵ are nothydrogen, e.g., of the formula (v-c).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (v) wherein R⁴ and R⁵ are hydrogen and R¹ and R² are nothydrogen, e.g., of the formula (v-d).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (v) wherein R² and R⁵ are hydrogen and R¹ and R⁴ are nothydrogen, e.g., of the formula (v-e).

In certain embodiments, R^(A) is a disubstituted pyridinyl of theformula (v) wherein R¹ and R⁵ are hydrogen and R² and R⁴ are nothydrogen, e.g., of the formula (v-f).

In certain embodiments, R^(A) is a 5,6-bicyclic heteroaryl.

For example, in certain embodiments, R^(A) is a 5,6-bicyclic heteroarylgroup of the formula (vi) (which is a subset of a group of the formula(ii-g)):

wherein R¹, R², R³ are as defined above and herein and R⁴ and R⁵ arejoined to form a 5-membered heteroaryl ring;

X, Y and Z are independently selected from CR^(A4), O, S, N, or NR^(A5);

each instance of R^(A4) is, independently, selected from hydrogen,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(A6), —ON(R^(A7))₂,—N(R^(A7))₂, —N(OR^(A6))R^(A8), —SH, —SR^(A6), —SSR^(A8), —C(═O)R^(A6),—CO₂H, —CHO, —C(OR^(A8))₂, —CO₂R^(A6), —OC(═O)R^(A6), —OCO₂R^(A6),—C(═O)N(R^(A7))₂, —OC(═O)N(R^(A7))₂, —NR^(A7)C(═O)R^(A6),—NR^(A7)CO₂R^(A6), —NR^(A7)C(═O)N(R^(A7))₂, —C(═NR^(A7))OR^(A6),—OC(═NR^(A7))R^(A6), —OC(═NR^(A7))OR^(A6), —C(═NR^(A7))N(R^(A7))₂,—OC(═NR^(A7))N(R^(A7))₂, —NR^(A7)C(═NR^(A7))N(R^(A7))₂,—C(═O)NR^(A7)SO₂R^(A6), —NR^(A7)SO₂R^(A6), —SO₂N(R^(A7))₂, —SO₂R^(A6),—SO₂OR^(A6), —OSO₂R^(A6), —S(═O)R^(A6), —OS(═O)R^(A6), —Si(R^(A6))₃,—OSi(R^(A6))₃, —C(═S)N(R^(A7))₂, —C(═O)SR^(A6), —C(═S)SR^(A6),—SC(═S)SR^(A6), —P(═O)₂R^(A6), —OP(═O)₂R^(A6), —P(═O)(R^(A6))₂,—OP(═O)(R^(A6))₂, —OP(═O)(OR^(A8))₂, —P(═O)₂N(R^(A7))₂,—OP(═O)₂N(R^(A7))₂, —P(═O)(NR^(A7))₂, —OP(═O)(NR^(A7))₂,—NR^(A7)P(═O)(OR^(A8))₂, —NR^(A7)P(═O)(NR^(A7))₂, —P(R^(A8))₂,—P(R^(A8))₃, —OP(R^(A8))₂, —OP(R^(A8))₃, —B(OR^(A8))₂, or—BR^(A6)(OR^(A8)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl;

each instance of R^(A6) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl;

each instance of R^(A5) and R^(A7) is, independently, selected fromhydrogen, —OH, —OR^(A6), —N(R^(A7))₂, —CN, —C(═O)R^(A6),—C(═O)N(R^(A7))₂, —CO₂R^(A6), —SO₂R^(A7), —C(═NR^(A3))OR^(A6),—C(═NR^(A7))N(R^(A7))₂, —SO₂N(R^(A3))₂, —SO₂R^(A6), —SO₂OR^(A8),—SOR^(A6), —C(═S)N(R^(A7))₂, —C(═O)SR^(A8), —C(═S) SR^(A),—P(═O)₂R^(A6), —P(═O)(R^(A6))₂, —P(═O)₂N(R^(A8))₂, —P(═O)(NR^(A8))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(A7) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring;

each instance of R^(A8) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, or two R^(A8) groups are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring; and

the dashed line represents a double or single bond.

In certain embodiments, R¹ is hydrogen. In certain embodiments, R² ishydrogen. In certain embodiments, R³ is hydrogen. In certainembodiments, R¹, R² and R³ are hydrogen.

In certain embodiments, R^(A) is a heteroaryl group of the formulae(vi-a) or (vi-b):

wherein R¹, R², R³ are as defined above and X and Z are independentlyselected from O, S and NR^(A5)

In certain embodiments, wherein R^(A) is a heteroaryl group of theformulae (vi-a) or (vi-b), X and Z are O (i.e., benzoxazolyl). Incertain embodiments, X and Z are S (i.e., benzthiazolyl). In certainembodiments, X and Z are NR^(A5) (i.e., imidazolyl).

In certain embodiments, R^(A) is a heteroaryl group of the formulae(vi-c) or (vi-d):

wherein R¹, R², R³ are as defined above and X is independently selectedfrom O, S and NRAS

In certain embodiments, wherein R^(A) is a heteroaryl group of theformulae (vi-c) or (vi-d), X is O (i.e., benzisoxazolyl). In certainembodiments, X is S (i.e., benzisothiazolyl). In certain embodiments, Xis NR^(A5) (i.e., indazolyl).

In certain embodiments, R^(A) is a heteroaryl group of the formulae(vi-e), (vi-f) or (vi-g):

wherein R¹, R², R³ and R^(A4) are as defined above and X, Y and Z areindependently selected from O, S and NR^(A5)

In certain embodiments, wherein R^(A) is a heteroaryl group of theformulae (vi-e), (vi-f) or (vi-g), Y is O (i.e., benzofuranyl orisobenzofuranyl). In certain embodiments, Y is S (i.e., benzothiophenylor isobenzothiophenyl). In certain embodiments, Y is NR^(A5) (i.e.,indolyl or isoindolyl).

In certain embodiments, R^(A) is a heteroaryl group of the formula(vi-h):

wherein R¹, R², R³ are as defined above and Y is independently selectedfrom O, S and NR^(A5).

In certain embodiments, wherein R^(A) is a heteroaryl group of theformula (vi-e), Y is O (i.e., benzoxadiazolyl). In certain embodiments,Y is S (i.e., benzthiadiazolyl). In certain embodiments, Y is NR^(A5)(i.e., benztriazolyl).

Group R^(B)

As described generally above, R^(B) is selected from C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl and 5-14 memberedheteroaryl; or R^(B) and R^(C) together with the nitrogen (N) atom towhich each is attached are joined to form a 5-14 membered ring.

In certain embodiments, R^(B) is selected from C₁₋₁₀ alkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl and 5-14 memberedheteroaryl.

In certain embodiments, R_(B) is an acyclic group, i.e., selected fromC₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl and 3-14 memberedheteroaliphatic. In certain embodiments, R^(B) is C₁₋₁₀ alkyl. Incertain embodiments, R^(B) is a substituted C₁₋₁₀ alkyl, e.g., a C₁₋₁₀aralkyl group. In certain embodiments, R^(B) is a C₁₋₂ aralkyl, e.g.,for example, a substituted or unsubstituted benzyl group (C₁ aralkyl) orsubstituted or unsubstituted phenylethyl group (C₂ aralkyl). In certainembodiments, R^(B) is a C₁₋₁₀ heteroaralkyl. In certain embodiments,R^(B) is alkenyl. In certain embodiments, R^(B) is alkynyl. In certainembodiments, R^(B) is 3-14 membered heteroaliphatic.

Alternatively, in certain embodiments, R^(B) is a cyclic group, i.e.,selected from C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryland 5-14 membered heteroaryl.

In certain embodiments, R^(B) is C₃₋₁₀ carbocyclyl or 3-14 memberedheterocyclyl.

In certain embodiments, R^(B) is C₃₋₁₀ carbocyclyl. Exemplarycarbocyclyl groups include, but are not limited to, cyclopropyl (C₃),cyclobutyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅), cyclohexyl (C₆),cyclohexenyl (C₆), cyclohexadienyl (C₆), cycloheptyl (C₇),cycloheptadienyl (C₇), cycloheptatrienyl (C₇) and cyclooctyl (C₈).

In certain embodiments, R^(B) is 3-14 membered heterocyclyl. Exemplaryheterocyclyl groups include, but are not limited to, azirdinyl,oxiranyl, thiorenyl, azetidinyl, oxetanyl, thietanyl, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl,dihydropyrrolyl, dioxolanyl, oxathiolanyl, dithiolanyl, piperidinyl,tetrahydropyranyl, dihydropyridinyl, thianyl, piperazinyl, morpholinyl,dithianyl, dioxanyl, azepanyl, oxepanyl thiepanyl, azocanyl, oxecanyland thiocanyl.

In certain embodiments, R^(B) is C₆₋₁₄ aryl or 5-14 membered heteroaryl.

In certain embodiments, R^(B) is C₆₋₁₄ aryl. Exemplary aryl groupsinclude, but are not limited to, phenyl, naphthyl and anthracyl. Incertain embodiments, R^(B) is phenyl (C₆ aryl). In certain embodiments,R^(B) is naphthyl (C₁₀ aryl).

In certain embodiments, R^(B) is 5-14 membered heteroaryl. In certainembodiments, R^(B) is 5-10 membered heteroaryl. In certain embodiments,R^(B) is 5-6 membered heteroaryl. In certain embodiments, R^(B) is a5,6-bicyclic heteroaryl. In certain embodiments, R^(B) is a 6,6-bicyclicheteroaryl.

In certain embodiments, R^(B) is a 5-membered heteroaryl group.Exemplary 5-membered heteroaryl groups include, but are not limited to,pyrrolyl, furanyl, thiophenyl, imidazolyl, pyrazolyl, oxazolyl,isoxazolyl, thiazolyl, isothiazolyl, triazolyl, oxadiazolyl,thiadiazolyl and tetrazolyl.

In certain embodiments, R^(B) is a 6-membered heteroaryl group.Exemplary 6-membered heteroaryl groups include, but are not limited to,pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl andtetrazinyl.

In certain embodiments, R^(B) is a 5,6-bicyclic heteroaryl group.Exemplary 5,6-bicyclic heteroaryl groups include, but are not limitedto, indolyl, isoindolyl, indazolyl, benztriazolyl, benzothiophenyl,isobenzothiophenyl, benzofuranyl, benzoisofuranyl, benzimidazolyl,benzoxazolyl, benzisoxazolyl, benzoxadiazolyl, benzthiazolyl,benzisothiazolyl, benzthiadiazolyl, indolizinyl, and purinyl.

In certain embodiments, R^(B) is a 6,6-bicyclic heteroaryl group.Exemplary 6,6-bicyclic heteroaryl groups include, but are not limitedto, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl, cinnolinyl,quinoxalinyl, phthalazinyl and quinazolinyl.

In certain embodiments, R^(B) is substituted with the group:

-L-R^(D)

wherein:

L is a covalent bond or a divalent C₁₋₁₀ hydrocarbon chain, wherein one,two or three methylene units of L are optionally and independentlyreplaced with one or more —O—, —S—, —NR^(B8)—, —(C═NR^(B8))—, —C(═O)—,—C(═S)—, —S(═O)—, —S(═O)₂—, divalent C₃₋₁₀ carbocyclyl, divalent 3-14membered heterocyclyl, divalent C₆₋₁₄ aryl or divalent 5-14 memberedheteroaryl group; and

R^(D) is selected from —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —C(═O)R^(B7),—CO₂H, —CHO, —C(OR^(B9))₂, —CO₂R^(B7), —OC(═O)R^(B7), —OCO₂R^(B7),—C(═O)N(R^(B8))₂, —OC(═O)N(R^(B8))₂, —NR^(B8)C(═O)R^(B7),—NR^(B8)CO₂R^(B7), —NR^(B8)C(═O)N(R^(B8))₂, —C(═NR^(B8))ORB,—OC(═NR^(B8))R^(B7), —OC(═NR^(B8))OR^(B7), —C(═NR^(B8))N(R^(B8))₂,—OC(═NR^(B8))N(R^(B8))₂, —NR^(B8)C(═NR^(B8))N(R^(B8))₂,—C(═O)NR^(B8)SO₂R^(B7), —NR^(B8)SO₂R^(B7), —SO₂N(R^(B8))₂, —SO₂R^(B7),—SO₂OR^(B7), —OSO₂R^(B7), —S(═O)R^(B7), —OS(═O)R^(B7), —C(═S)N(R^(B8))₂,—C(═O)SR^(B7), —C(═S)SR^(B7), —SC(═S)SR^(B7), —P(═O)₂R^(B7),—OP(═O)₂R^(B7), —P(═O)(R^(B7))₂, —OP(═O)(R^(B7))₂, —OP(═O)(OR^(B9))₂,—P(═O)₂N(R^(B8))₂, —OP(═O)₂N(R^(B8))₂, —P(═O)(NR^(B8))₂,—OP(═O)(NR^(B8))₂, —NR^(B8)P(═O)(OR^(B9))₂, —NR^(B8)P(═O)(NR^(B8))₂,—B(OR^(B9))₂, —BR^(B7)(OR^(B9)), and tetrazolyl;

each instance of R^(B7) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl;

each instance of R^(B8) is, independently, selected from hydrogen, —OH,—OR^(B7), —N(R^(B9))₂, —CN, —C(═O)R^(B7), —C(═O)N(R^(B9))₂, —CO₂R^(B7),—SO₂R^(B7), —C(═NR^(B9))OR^(B7), —C(═NR^(B9))N(R^(B9))₂, —SO₂N(R^(B9))₂,—SO₂R^(B9), —SO₂OR^(B9), —SOR^(B7), —C(═S)N(R^(B9))₂, —C(═O)SR^(B9),—C(═S)SR^(B9), —P(═O)₂R^(B7), —P(═O)(R^(B7))₂, —P(═O)₂N(R^(B9))₂,—P(═O)(NR^(B9))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B8)groups are joined to form a 3-14 membered heterocyclyl or a 5-14membered heteroaryl ring; and

each instance of R^(B9) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, or two R^(B9) groups are joined toform a 3-14 membered heterocyclyl or a 5-14 membered heteroaryl ring.

In certain embodiments, L is a covalent bond.

In certain embodiments, L is a divalent C₁₋₁₀ hydrocarbon chain, whereinone, two or three methylene units of L are optionally and independentlyreplaced with one or more —O—, —S—, —NR^(B8)—, —(C═NR^(B8))—, —C(═O)—,—C(═S)—, —S(═O)—, —S(═O)₂—, divalent carbocyclyl, divalent heterocyclyl,divalent aryl or divalent heteroaryl group.

In certain embodiments, L is a divalent C₁₋₁₀ hydrocarbon chain, whereinone, two or three methylene units of L are optionally and independentlyreplaced with one or more —O—, —S—, —NR^(B8)—, —(C═NR^(B8))—, —C(═O)—,—C(═S)—, —S(═O)—, —S(═O)₂—, divalent C₃₋₁₀ carbocyclyl, divalent 3-14membered heterocyclyl, divalent C₆₋₁₄ aryl or divalent 5-14 memberedheteroaryl group.

As generally described above, R^(D) is selected from the groupconsisting of —CN, —NO₂, —SO₂H, —SO₃H, —C(═O)R^(B7), —CO₂H, —CHO,—C(OR^(B9))₂, —CO₂R^(B7), —OC(═O)R^(B7), —OCO₂R^(B7), —C(═O)N(R^(B8))₂,—OC(═O)N(R^(B8))₂, —NR^(B8)C(═O)R^(B7), —NR^(B8)CO₂R^(B7)—,—NR^(B8)C(═O)N(R^(B8))₂, —C(═NR^(B8))OR^(B7), —OC(═NR^(B8))R^(B7),—OC(═NR^(B8))OR^(B7), —C(═NR^(B8))N(R^(B8))₂, —OC(═NR^(B8))N(R^(B8))₂,—NR^(B8)C(═NR^(B8))N(R^(B8))₂, —C(═O)NR^(B8)SO₂R^(B7),—NR^(B8)SO₂R^(B7), —SO₂N(R^(B8))₂, —SO₂R^(B7), —SO₂OR^(B7), —OSO₂R^(B7),—S(═O)R^(B7), —OS(═O)R^(B7), —C(═S)N(R^(B8))₂, —C(═O)SR^(B7),—C(═S)SR^(B7), —SC(═S)SR^(B7), —P(═O)₂R^(B7), —OP(═O)₂R^(B7),—P(═O)(R^(B7))₂, —OP(═O)(R^(B7))₂, —OP(═O)(OR^(B9))₂, —P(═O)₂N(R^(B8))₂,—OP(═O)₂N(R^(B8))₂, —P(═O)(NR^(B8))₂, —OP(═O)(NR^(B8))₂,—NR^(B8)P(═O)(OR^(B9))₂, —NR^(B8)P(═O)(NR^(B8))₂, —B(OR^(B9))₂,—BR^(B7)(OR^(B9)) and tetrazolyl.

However, in certain embodiments, R^(D) is not —CO₂R^(B7) (e.g., CO₂Me,CO₂Et, CO₂nPr, CO₂iPr, or CO₂tBu), but can be selected from any of theother substituents listed above. In certain embodiments, R^(D) is not—C(═O)R^(B7)), but can be selected from any of the other substituentslisted above. In certain embodiments, R^(D) is not —CHO), but can beselected from any of the other substituents listed above. In certainembodiments, R^(D) is not —C(OR^(B9))₂), but can be selected from any ofthe other substituents listed above. In certain embodiments, R^(D) isnot —CN), but can be selected from any of the other substituents listedabove. In certain embodiments, R^(D) is not —NO₂), but can be selectedfrom any of the other substituents listed above. In certain embodiments,R^(D) is not any one of —SO₂H, —SO₃H, —SO₂N(R^(B8))₂, —NR^(B8)SO₂R^(B7),—SO₂R^(B7), —SO₂OR^(B7), —OSO₂R^(B7), —S(═O)R^(B7) or —OS(═O)R^(B7)),but can be selected from any of the other substituents listed above. Incertain embodiments, R^(D) is not any one of —OC(═O)R^(B7), —OCO₂R^(B7),—OC(═O)N(R^(B8))₂, —NR^(B8)C(═O)R^(B7), —NR^(B8)CO₂R^(B7),—NR^(B8)C(═O)N(R^(B8))₂, —OC(═NR^(B8))R^(B7), —OC(═NR^(B8))OR^(B7),—OC(═NR^(B8))N(R^(B8))₂ or —NR^(B8)C(═NR^(B8))N(R^(B8))₂), but can beselected from any of the other substituents listed above. In certainembodiments, R^(D) is not any one of —C(═S)N(R^(B8))₂, —C(═O)SR^(B7),—C(═S)SR^(B7) or —SC(═S)SR^(B7)), but can be selected from any of theother substituents listed above. In certain embodiments, R^(D) is notany one of —P(═O)₂R^(B7), —OP(═O)₂R^(B7), —P(═O)(R^(B7))₂,—OP(═O)(R^(B7))₂, —OP(═O)(OR^(B9))₂, —P(═O)₂N(R^(B8))₂,—OP(═O)₂N(R^(B8))₂, —P(═O)(NR^(B8))₂, —OP(═O)(NR^(B8))₂,—NR^(B8)P(═O)(OR^(B9))₂ or —NR^(B8)P(═O)(NR^(B8))₂), but can be selectedfrom any of the other substituents listed above. In certain embodiments,R^(D) is not any one of —B(OR^(B9))₂ or —BR^(B7)(OR^(B9))), but can beselected from any of the other substituents listed above. In certainembodiments, R^(D) is not tetrazolyl), but can be selected from any ofthe other substituents listed above.

In certain embodiments, R^(D) is selected from —CN, —NO₂, —SO₂H, —SO₃H,—C(═O)R^(B7), —CO₂H, —CHO, —CO₂R^(B7), —C(═O)N(R^(B8))₂,—C(═NR^(B8))OR^(B7), —C(═NR^(B8))N(R^(B8))₂, —C(═O)NR^(B8)SO₂R^(B7),—SO₂N(R^(B8))₂, —SO₂R^(B7), —SO₂OR^(B7), —S(═O)R^(B7), —C(═S)N(R^(B8))₂,—C(═O)SR^(B7), —C(═S)SR^(B7), —P(═O)₂R^(B7), —P(═O)(R^(B7))₂,—P(═O)₂N(R^(B8))₂, —P(═O)(NR^(B8))₂, —B(OR^(B9))₂, —BR^(B7)(OR^(B9)) andtetrazolyl. In certain embodiments, L is a covalent bond.

In certain embodiments, R^(D) is selected from —C(═O)R^(B7), —CO₂H,—CHO, —CO₂R^(B7), —C(═O)N(R^(B8))₂, —C(═NR^(B8))OR^(B7),—C(═NR^(B8))N(R^(B8))₂, —C(═O)NR^(B8)SO₂R^(B7), —C(═S)N(R^(B8))₂,—C(═O)SR^(B7) and —C(═S)SR^(B7). In certain embodiments, L is a covalentbond.

In certain embodiments, R^(D) is selected from —C(═O)R^(B7), —CO₂H,—CHO, and —CO₂R^(B7). In certain embodiments, L is a covalent bond.

In certain embodiments, R^(D) is —CO₂H. In certain embodiments, L is acovalent bond.

In certain embodiments, wherein R^(B) is substituted with -L-R^(D),R^(B) is further substituted with the group:

—R^(E)

wherein:

R^(E) is selected from halogen, —OH, —OR^(B10), —ON(R^(B11))₂,—N(R^(B11))₂, —N(OR^(B12))R^(B12), —SH, —SR^(B10), —SSR^(B12),—OC(═O)R^(B10), —OCO₂R^(B10), —OC(═O)N(R^(B11))₂, —NR^(B11)C(═O)R^(B10),—NR^(B11)CO₂R^(B10), —NR^(B11)C(═O)N(R^(B11))₂, —OC(═NR^(B11))R^(B10),—OC(═NR^(B11))OR^(B10), —OC(═NR^(B11))N(R^(B11))₂,—NR^(B11)C(═NR^(B11))N(R^(B11))₂, —NR^(B11)SO₂R^(B10), —OSO₂R^(B10),—OS(═O)R^(B10), —Si(R^(B10))₃, —OSi(R^(B10))₃, —SC(S)SR^(B10),—OP(═O)₂R^(B10), —OP(═O)(R^(B10))₂, —OP(═O)(OR^(B12))₂,—OP(═O)₂N(R^(B11))₂, —OP(═O)(NR^(B11))₂, —NR^(B11)P(═O)(OR^(B12))₂,—NR^(B11)P(═O)(NR^(B11))₂, —P(R^(B12))₂, —P(R^(B12))₃, —OP(R^(B12))₂,—OP(R^(B12))₃, 3-14 membered heterocyclyl and 5-14 membered heteroaryl,wherein the point of attachment of the 3-14 membered heterocyclyl or5-14 membered heteroaryl group is on a nitrogen atom;

each instance of R^(B10) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl;

each instance of R^(B11) is, independently, selected from hydrogen, —OH,—OR^(B10), —N(R^(B12))₂, —CN, —C(═O)R^(B10), —C(═O)N(R^(B12))₂,—CO₂R^(B10), —SO₂R^(B10), —C(═NR^(B12))OR^(B10),—C(═NR^(B12))N(R^(B12))₂, —SO₂N(R^(B12))₂, —SO₂R^(B12), —SO₂OR^(B12),—SOR^(B10), —C(═S)N(R^(B12))₂, —C(═O)SR^(B12), —C(═S)SR^(B12),—P(═O)₂R^(B10), —P(═O)(R^(B10))₂, —P(═O)₂N(R^(B12))₂, —P(═O)(NR^(B12))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B11) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring; and

each instance of R^(B12) is, independently, selected from hydrogen,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B12) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring.

In certain embodiments, R^(E) is selected from halogen, —OH, —OR^(B10),—ON(R^(B11))₂, —N(R^(B11))₂, —N(OR^(B12))R^(B12), —SH, —SR^(B10),—SSR^(B12), —Si(R^(B10))₃, —OSi(R^(B10))₃, —P(R^(B12))₂, —P(R^(B12))₃,—OP(R^(B12))₂, —OP(R^(B12))₃, 3-14 membered heterocyclyl and 5-14membered heteroaryl, wherein the point of attachment of the 3-14membered heterocyclyl or 5-14 membered heteroaryl group is on a nitrogenatom.

In certain embodiments, R^(E) is selected from halogen, —OH, —OR^(B10),—N(R^(B11))₂, 3-14 membered heterocyclyl and 5-14 membered heteroaryl,wherein the point of attachment of the 3-14 membered heterocyclyl or5-14 membered heteroaryl group is on a nitrogen atom.

In certain embodiments, R^(E) is selected from halogen, —OR^(B10) and—N(R^(B11))₂. In certain embodiments, R^(E) is halogen. In certainembodiments, R^(E) is —OR^(B10). In certain embodiments, R^(E) is—N(R^(B11))₂.

In certain embodiments, -L-R^(D) and —R^(E) are vicinal R^(B)substituents (i.e., attached to two adjacent atoms on the group R^(B);e.g., ortho to each other). In certain embodiments, -L-R^(D) and —R^(E)are ortho to each other.

In certain embodiments, -L-R^(D) and —R^(E) are not vicinal R^(B)substituents (i.e., not attached to two adjacent atoms on the groupR^(B); e.g., meta orpara to each other). In certain embodiments,-L-R^(D) and —R^(E) are meta to each other. In certain embodiments,-L-R^(D) and —R^(E) are para to each other.

In certain embodiments, the R^(B) is a group of the formula (vii):

wherein each group W—R⁶, W—R⁷, W—R⁸, W—R⁹, and W—R¹⁰ independentlyrepresents either a nitrogen atom (N) or C—R⁶, C—R⁷, C—R⁸, C—R⁹, orC—R¹⁰, respectively; and

wherein R⁶, R⁷, R⁸, R⁹ and R¹⁰ are independently selected from the groupconsisting of hydrogen, halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH,—OR^(B1), —ON(R^(B2))₂, —N(R^(B2))₂, —N(OR^(B3))R^(B3), —SH, —SR^(B1),—SSR^(B3), —C(═O)R^(B1), —CO₂H, —CHO, —C(OR^(B3))₂, —CO₂R^(B1),—OC(═O)R^(B1), —OCO₂R^(B1), —C(═O)N(R^(B2))₂, —OC(═O)N(R^(B2))₂,—NR^(B2)C(═O)R^(B1), —NR^(B2)CO₂R^(B1), —NR^(B2)C(═O)N(R^(B2))₂,—C(═NR^(B2))OR^(B1), —OC(═NR^(B2))R^(B1), —OC(═NR^(B2))OR^(B1),—C(═NR^(B2))N(R^(B2))₂, —OC(═NR^(B2))N(R^(B2))₂,—NR^(B2)C(═NR^(B2))N(R^(B2))₂, —C(═O)NR^(B2)SO₂R^(B1),—NR^(B2)SO₂R^(B1), —SO₂N(R^(B2))₂, —SO₂R^(B1), —SO₂OR^(B1), —OSO₂R^(B1),—S(═O)R^(B1), —OS(═O)R^(B1), —Si(R^(B1))₃,—OSi(R^(B1))₃—C(═S)N(R^(B2))₂, —C(═O)SR^(B1), —C(═S)SR^(B1),—SC(S)SR^(B1), —P(═O)₂R^(B1), —OP(═O)₂R^(B1), —P(═O)(R^(B1))₂,—OP(═O)(R^(B1))₂, —OP(═O)(OR^(B3))₂, —P(═O)₂N(R^(B2))₂,—OP(═O)₂N(R^(B2))₂, —P(═O)(NR^(B2))₂, —OP(═O)(R^(B2))₂,—NR^(B2)P(═O)(OR^(B3))₂, —NR^(B2)P(═O)(NR^(B2))₂, —P(R^(B3))₂,—P(R^(B3))₃, —OP(R^(B3))₂, —OP(R^(B3))₃, —B(OR^(B3))₂, or—BR^(B1)(OR^(B3)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, 5-14 membered heteroaryl, -L-R^(D) and —R^(E);or one or more of R⁶ and R⁷, R⁷ and R⁸, R⁸ and R⁹ or R⁹ and R¹⁰ arejoined to form a C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl or 5-14 membered heteroaryl ring; or R¹⁰ and R^(C) are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring;

each instance of R^(B1) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl;

each instance of R^(B2) is, independently, selected from hydrogen, —OH,—OR^(B1), —N(R^(B3))₂, —CN, —C(═O)R^(B1), —C(═O)N(R^(B3))₂, —CO₂R^(B1),—SO₂R^(B1), —C(═NR^(B3))OR^(B1), —C(═NR^(B3))N(R^(B3))₂, —SO₂N(R^(B3))₂,—SO₂R^(B3), —SO₂OR^(B3), —SOR^(B1), —C(═S)N(R^(B3))₂, —C(═O)SR^(B3),—C(═S)SR^(B3), —P(═O)₂R^(B1), —P(═O)(R^(B1))₂, —P(═O)₂N(R^(B3))₂,—P(═O)(NR^(B3))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B2)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring;

each instance of R^(B3) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, or two R^(B3) groups are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring;

and L, R^(D) and R^(E) are as defined above and herein.

As used herein, when one or more of R⁶, R⁷, R⁸, R⁹ and R¹⁰ is referredto as “not hydrogen”, it is meant that one or more of R⁶, R⁷, R⁸, R⁹ andR¹⁰ is independently selected from the group consisting of halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(B1), —ON(R^(B2))₂, —N(R^(B2))₂,—N(OR^(B3))R^(B3), —SH, —SR^(B1), —SSR^(B3), —C(═O)R^(B1), —CO₂H, —CHO,—C(OR^(B3))₂, —CO₂R^(B1), —OC(═O)R^(B1), —OCO₂R^(B1), —C(═O)N(R^(B2))₂,—OC(═O)N(R^(B2))₂, —NR^(B2)C(═O)R^(B1), —NR^(B2)CO₂R^(B1),—NR^(B2)C(═O)N(R^(B2))₂, —C(═NR^(B2))OR^(B1), —OC(═NR^(B2))R^(B1),—OC(═NR^(B2))OR^(B1), —C(═NR^(B2))N(R^(B2))₂, —OC(═NR^(B2))N(R^(B2))₂,—NR^(B2)C(═NR^(B2))N(R^(B2))₂, —C(═O)NR^(B2)SO₂R^(B1),—NR^(B2)SO₂R^(B1), —SO₂N(R^(B2))₂, —SO₂R^(B1), —SO₂OR^(B1), —OSO₂R^(B1),—S(═O)R^(B1), —OS(═O)R^(B1), —Si(R^(B1))₃,—OSi(R^(B1))₃—C(═S)N(R^(B2))₂, —C(═O)SR^(B1), —C(═S)SR^(B1),—SC(═S)SR^(B1), —P(═O)₂R^(B1), —OP(═O)₂R^(B1), —P(═O)(R^(B1))₂,—OP(═O)(R^(B1))₂, —OP(═O)(OR^(B3))₂, —P(═O)₂N(R^(B2))₂,—OP(═O)₂N(R^(B2))₂, —P(═O)(NR^(B2))₂, —OP(═O)(NR^(B2))₂,—NR^(B2)P(═O)(OR^(B3))₂, —NR^(B2)P(═O)(NR^(B2))₂, —P(R^(B3))₂,—P(R^(B3))₃, —OP(R^(B3))₂, —OP(R^(B3))₃, —B(OR^(B3))₂,—BR^(B1)(OR^(B3)), -L-R^(D), —R^(E), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl,C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl; or wherein one or more of R⁶ and R⁷, R⁷ and R⁸, R⁸ and R⁹ orR⁹ and R¹⁰ are joined to form a C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl or 5-14 membered heteroaryl ring, or whereinR¹⁰ and R^(C) are joined to form a 3-14 membered heterocyclyl or 5-14membered heteroaryl ring.

In certain embodiments, at least one of R⁶, R⁷, R⁸, R⁹, and R¹⁰ is thegroup -L-R^(D) as defined above and herein. In certain embodiments, atleast one of R⁶, R⁷, R⁸, R⁹, and R¹⁰ is the group —R^(E) as definedherein.

In certain embodiments, the group of formula (vii) represents a C₆₋₁₄aryl or a 6-14 membered heteroaryl group. In certain embodiments, thegroup of formula (vii) represents a 6-14 membered heteroaryl group. Incertain embodiments, the group of formula (vii) represents a C₆₋₁₄ arylgroup. In certain embodiments, the group of formula (vii) represents aphenyl group.

In certain embodiments, W—R⁶, W—R⁷, W—R⁸, W—R⁹, and W—R¹⁰ representC—R⁶, C—R⁷, C—R⁸, C—R⁹, or C—R¹⁰, respectively. For example, in certainembodiments, R^(B) is a group of the formula (viii):

wherein R⁶, R⁷, R⁸, R⁹ and R¹⁰ are as defined above and herein.

In certain embodiments, at least one of R⁶, R⁷, R⁸, R⁹, and R¹⁰ is thegroup -L-R^(D) as defined above and herein. In certain embodiments, atleast one of R⁶, R⁷, R⁸, R⁹, and R¹⁰ is the group —R^(E) as definedherein.

In certain embodiments, the group of the formula (viii) represents aC₆₋₁₄ aryl group. In certain embodiments, the C₆₋₁₄ aryl group of theformula (viii) represents a phenyl group.

In certain embodiments, R^(B) is a monosubstituted, disubstituted ortrisubstituted group of the formula (viii). In certain embodiments,R^(B) is a monosubstituted or disubstituted group of the formula (viii).

In certain embodiments, R^(B) is a monosubstituted group of the formula(viii).

For example, in certain embodiments, R^(B) is an ortho-substituted groupof formula (viii), e.g., wherein R⁶-R⁹ are hydrogen, and R¹⁰ is nothydrogen, e.g., of the formula (viii-a).

In certain embodiments, R^(B) is a meta-substituted group of the formula(viii), e.g., wherein R⁶-R⁸ and R¹⁰ are hydrogen and R⁹ is not hydrogen,e.g., of the formula (viii-b).

In certain embodiments, R^(B) is a para-substituted group of the formula(viii), e.g., wherein R⁶, R⁷, R⁹ and R¹⁰ are hydrogen and R⁸ is nothydrogen, e.g., of the formula (viii-c).

In certain embodiments, R^(B) is a disubstituted group of the formula(viii).

For example, in certain embodiments, R^(B) is a 2,6-disubstituted groupof the formula (viii), e.g., wherein R⁷, R⁸ and R⁹ are hydrogen, and R⁶and R¹⁰ are not hydrogen, e.g., of the formula (viii-d).

In certain embodiments, R^(B) is a 2,5-disubstituted group of theformula (viii), e.g., wherein R⁶, R⁸ and R⁹ are hydrogen, and R⁷ and R¹⁰are not hydrogen, e.g., of the formula (viii-e).

In certain embodiments, R^(B) is a 2,4-disubstituted group of theformula (viii), e.g., wherein R⁶, R⁷ and R⁹ are hydrogen, and R⁸ and R¹⁰are not hydrogen, e.g., of the formula (viii-f).

In certain embodiments, R^(B) is a 2,3-disubstituted group of formula(viii), e.g., wherein R⁶, R⁷ and R⁸ are hydrogen, and R⁹ and R¹⁰ are nothydrogen, e.g., of the formula (viii-g).

In certain embodiments, R^(B) is a 3,4-disubstituted group of theformula (viii), e.g., wherein R⁶, R⁷ and R¹⁰ are hydrogen, and R⁸ and R⁹are not hydrogen, e.g., of the formula (viii-h).

In certain embodiments, R^(B) is a 3,5-disubstituted group of theformula (viii), e.g., wherein R⁶, R⁷ and R¹⁰ are hydrogen, and R⁷ and R⁹are not hydrogen, e.g., of the formula (viii-i).

In certain embodiments, R^(B) is a trisubstituted group of the formula(viii).

For example, in certain embodiments, R^(B) is a 2,4,6-trisubstitutedgroup of formula (viii), e.g., wherein R⁷ and R⁹ are hydrogen, and R⁶,R⁸ and R¹⁰ are not hydrogen, e.g., of the formula (viii-j).

In certain embodiments, R^(B) is a 2,3,6-trisubstituted group of theformula (viii), e.g., wherein R² and R³ are hydrogen, and R¹, R⁴ and R⁵are not hydrogen, e.g., of the formula (viii-k).

In certain embodiments, R^(B) is a 2,4,5-trisubstituted group of theformula (viii), e.g., wherein R⁸ and R⁹ are hydrogen, and R⁶, R⁷ and R¹⁰are not hydrogen, e.g., of the formula (viii-1).

In certain embodiments, R^(B) is a 2,3,4-trisubstituted group of theformula (viii), e.g., wherein R⁶ and R⁹ are hydrogen, and R⁷, R⁸ and R¹⁰are not hydrogen, e.g., of the formula (viii-m).

In certain embodiments, R^(B) is a 3,4,5-trisubstituted group of theformula (viii), e.g., wherein R⁶ and R¹⁰ are hydrogen, and R⁷, R⁸ and R⁹are not hydrogen, e.g., of the formula (viii-n).

In certain embodiments, R^(B) is heteroaryl selected from a 5-6-memberedheteroaryl, a 5,6-bicyclic heteroaryl, or a 6,6-bicyclic heteroaryl.

In certain embodiments, R^(B) is a 6-membered heteroaryl. In certainembodiments, R^(A) is a 6-membered heteroaryl selected from pyridinyl.In certain embodiments, R^(B) is 2-pyridinyl, 3-pyridinyl or4-pyridinyl.

In certain embodiments, R^(B) is a 2-pyridinyl wherein W—R⁶ is N, andW—R⁷, W—R⁸, W—R⁹, and W—R¹⁰ are C—R⁷, C—R⁸, C—R⁹ and C—R¹⁰,respectively, e.g., of the formula (ix).

In certain embodiments, R^(B) is a 3-pyridinyl wherein W—R⁷ is N, andW—R⁶, W—R⁸, W—R⁹, and W—R¹⁰ are C—R⁶, C—R⁸, C—R⁹ and C—R¹⁰,respectively, e.g., of the formula (x).

In certain embodiments, R^(B) is a 4-pyridinyl wherein W—R⁸ is N, andW—R⁶, W—R⁷, W—R⁹, and W—R¹⁰ are C—R⁶, C—R⁷, C—R⁹ and C—R¹⁰,respectively, e.g., of the formula (xi).

wherein R⁶, R⁷, R⁸, R⁹ and R¹⁰ are as defined above and herein.

In certain embodiments, at least one of R⁶, R⁷, R⁸, R⁹, and R¹⁰ is thegroup -L-R^(D) as defined above and herein. In certain embodiments, atleast one of R⁶, R⁷, R⁸, R⁹, and R¹⁰ is the group —R^(E) as definedherein.

In certain embodiments, R^(B) is a monosubstituted or disubstitutedpyridinyl.

In certain embodiments, R^(B) is a monosubstituted pyridinyl.

In certain embodiments, R^(B) is a monosubstituted pyridinyl of theformula (ix) wherein R⁸, R⁹, R¹⁰ are hydrogen and R⁷ is not hydrogen,e.g., of the formula (ix-a).

In certain embodiments, R^(B) is a monosubstituted pyridinyl of theformula (ix) wherein R⁷, R⁹, R¹⁰ are hydrogen and R⁸ is not hydrogen,e.g., of the formula (ix-b).

In certain embodiments, R^(B) is a monosubstituted pyridinyl of theformula (ix) wherein R⁷, R⁸, R¹⁰ are hydrogen and R⁹ is not hydrogen,e.g., of the formula (ix-c).

In certain embodiments, R^(B) is a monosubstituted pyridinyl of theformula (ix) wherein R⁷, R⁸, R⁹ are hydrogen and R¹⁰ is not hydrogen,e.g., of the formula (ix-d).

In certain embodiments, R^(B) is a monosubstituted pyridinyl of theformula (x) wherein R⁸, R⁹, R¹⁰ are hydrogen and R⁶ is not hydrogen,e.g., of the formula (x-a).

In certain embodiments, R^(B) is a monosubstituted pyridinyl of theformula (x) wherein R⁶, R⁹, R¹⁰ are hydrogen and R⁸ is not hydrogen,e.g., of the formula (x-b).

In certain embodiments, R^(B) is a monosubstituted pyridinyl of theformula (x) wherein R⁶, R⁸, R¹⁰ are hydrogen and R⁹ is not hydrogen,e.g., of the formula (x-c).

In certain embodiments, R^(B) is a monosubstituted pyridinyl of theformula (x) wherein R⁶, R⁸, R⁹ are hydrogen and R¹⁰ is not hydrogen,e.g., of the formula (x-d).

In certain embodiments, R^(B) is a monosubstituted pyridinyl of theformula (xi) wherein R⁶, R⁷, R⁹ are hydrogen and R¹⁰ is not hydrogen,e.g., of the formula (xi-a).

In certain embodiments, R^(B) is a monosubstituted pyridinyl of theformula (v) wherein R⁶, R⁷, R¹⁰ are hydrogen and R⁹ is not hydrogen,e.g., of the formula (xi-b).

In certain embodiments, R^(B) is a disubstituted pyridinyl.

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (ix) wherein R⁸ and R⁹ are hydrogen and R⁷ and R¹⁰ are nothydrogen, e.g., of the formula (ix-e).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (ix) wherein R⁷ and R⁹ are hydrogen and R⁸ and R¹⁰ are nothydrogen, e.g., of the formula (ix-f).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (ix) wherein R⁷ and R⁸ are hydrogen and R⁹ and R¹⁰ are nothydrogen, e.g., of the formula (ix-g).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (ix) wherein R⁸ and R¹⁰ are hydrogen and R⁷ and R⁹ are nothydrogen, e.g., of the formula (ix-h).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (ix) wherein R⁹ and R¹⁰ are hydrogen and R⁷ and R⁸ are nothydrogen, e.g., of the formula (ix-i).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (ix) wherein R⁷ and R¹⁰ are hydrogen and R⁸ and R⁹ are nothydrogen, e.g., of the formula (ix-j).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (x) wherein R⁸ and R⁹ are hydrogen and R⁶ and R¹⁰ are nothydrogen, e.g., of the formula (x-e).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (x) wherein R⁸ and R¹⁰ are hydrogen and R⁶ and R⁹ are nothydrogen, e.g., of the formula (x-f).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (x) wherein R⁹ and R¹⁰ are hydrogen and R⁶ and R⁸ are nothydrogen, e.g., of the formula (x-g).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (x) wherein R⁶ and R⁹ are hydrogen and R⁸ and R¹⁰ are nothydrogen, e.g., of the formula (x-h).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (x) wherein R⁶ and R¹⁰ are hydrogen and R⁸ and R⁹ are nothydrogen, e.g., of the formula (x-i).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (x) wherein R⁶ and R⁸ are hydrogen and R⁹ and R¹⁰ are nothydrogen, e.g., of the formula (x-j).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (xi) wherein R⁷ and R⁹ are hydrogen and R⁶ and R¹⁰ are nothydrogen, e.g., of the formula (xi-c).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (xi) wherein R⁶ and R⁷ are hydrogen and R⁹ and R¹⁰ are nothydrogen, e.g., of the formula (xi-d).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (xi) wherein R⁶ and R⁸ are hydrogen and R⁷ and R¹⁰ are nothydrogen, e.g., of the formula (xi-e).

In certain embodiments, R^(B) is a disubstituted pyridinyl of theformula (xi) wherein R⁶ and R¹⁰ are hydrogen and R⁷ and R⁹ are nothydrogen, e.g., of the formula (xi-f).

In certain embodiments, R^(B) is C₅₋₁₀ carbocyclyl or 5-10 memberedheterocyclyl of the formula (xii):

wherein:

X is N, —NR³⁰, O, S or CR³¹R³²;

p is 0, 1 or 2;

each instance of R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹, R³¹ andR³² is independently selected from hydrogen, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OR^(B1), —ON(R^(B2))₂, —N(R^(B2))₂,—N(OR^(B3))R^(B3), —SH, —SR^(B1), —SSR^(B3), —C(═O)R^(B1), —CO₂H, —CHO,—C(OR^(B3))₂, —CO₂R^(B1), —OC(═O)R^(B1), —OCO₂R^(B1), —C(═O)N(R^(B2))₂,—OC(═O)N(R^(B2))₂, —NR^(B2)C(═O)R^(B1), —NR^(B2)CO₂R^(B1),—NR^(B2)C(═O)N(R^(B2))₂, —C(═NR^(B2))OR^(B1), —OC(═NR^(B2))R^(B1),—OC(═NR^(B2))OR^(B1), —C(═NR^(B2))N(R^(B2))₂, —OC(═NR^(B2))N(R^(B2))₂,—NR^(B2)C(═NR^(B2))N(R^(B2))₂, —C(═O)NR^(B2)SO₂R^(B1),—NR^(B2)SO₂R^(B1), —SO₂N(R^(B2))₂, —SO₂R^(B1), —SO₂OR^(B1), —OSO₂R^(B1),—S(═O)R^(B1), —OS(═O)R^(B1), —Si(R^(B1))₃,—OSi(R^(B1))₃—C(═S)N(R^(B2))₂, —C(═O)SR^(B1), —C(═S)SR^(B1),—SC(═S)SR^(B1), —P(═O)₂R^(B1), —OP(═O)₂R^(B1), —P(═O)(R^(B1))₂,—OP(═O)(R^(B1))₂, —OP(═O)(OR^(B3))₂, —P(═O)₂N(R^(B2))₂,—OP(═O)₂N(R^(B2))₂, —P(═O)(NR^(B2))₂, —OP(═O)(NR^(B2))₂,—NR^(B2)P(═O)(OR^(B3))₂, —NR^(B2)P(═O)(NR^(B2))₂, —P(R^(B3))₂,—P(R^(B3))₃, —OP(R^(B3))₂, —OP(R^(B3))₃, —B(OR^(B3))₂, or—BR^(B1)(OR^(B3)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, 5-14 membered heteroaryl, -L-R^(D) and —R^(E);or one or more of R²⁹ and R²¹, R²² and R²³, R²⁴ and R³¹, R³² and R²⁵,R²⁶ and R²⁷, R²⁸ and R²⁹, or R²⁶ and R²⁹, are joined to form a doublebond or a C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl or5-14 membered heteroaryl ring; optionally wherein X is N, then N and R²³or N and R²⁵ are joined to form a double bond;

R³⁰ is selected from hydrogen, —OH, —OR^(B1), —N(R^(B3))₂, —CN,—C(═O)R^(B1), —C(═O)N(R^(B3))₂, —CO₂R^(B1), —SO₂R^(B1),—C(═NR^(B3))OR^(B1), —C(═NR^(B3))N(R^(B3))₂, —SO₂N(R^(B3))₂, —SO₂R^(B3),—SO₂OR^(B3), —S(═O)R^(B1), —C(═S)N(R^(B3))₂, —C(═O)SR^(B3),—C(═S)SR^(B3), —P(═O)₂R^(B1), —P(═O)(R^(B1))₂, —P(═O)₂N(R^(B3))₂,—P(═O)(NR^(B3))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, optionallywherein R²⁴ and R³⁰ or R³⁰ and R²⁵ are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring;

wherein:

each instance of R^(B1) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl;

each instance of R^(B2) is, independently, selected from hydrogen, —OH,—OR^(B1), —N(R^(B3))₂, —CN, —C(═O)R^(B1), —C(═O)N(R_(B3))₂, —CO₂R^(B1),—SO₂R^(B1), —C(═NR^(B3))OR^(B1), —C(═NR^(B3))N(R^(B3))₂, —SO₂N(R^(B3))₂,—SO₂R^(B3), —SO₂OR^(B3), —SOR^(B1), —C(═S)N(R_(B3))₂, —C(═O)SR^(B3),—C(═S)SR^(B3), —P(═O)₂R^(B1), —P(═O)(R^(B1))₂, —P(═O)₂N(R^(B3))₂,—P(═O)(NR^(B3))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B2)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring;

each instance of R^(B3) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, or two R^(B3) groups are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring;

and L, R^(D) and R^(E) are as defined above and herein.

In certain embodiments, at least one of R²¹, R²², R²³, R²⁴, R²⁵, R²⁶,R²⁷, R²⁸, R²⁹, R³⁰, R³¹, and R³² is the group -L-R^(D) as defined aboveand herein. In certain embodiments, at least one of R²¹, R²², R²³, R²⁴,R²⁵, R²⁶, R²⁷, R²⁸, R²⁹, R³⁰, R³¹, and R³² is selected from the group—R^(E) as defined herein.

In certain embodiments, p is 0. In certain embodiments, p is 1. Incertain embodiments, p is 2.

In certain embodiments, X is N. In certain embodiments, X is NR³⁰. Incertain embodiments, X is O. In certain embodiments, X is S. In certainembodiments, X is CR³¹R³²

In certain embodiments, R^(B) is C₅₋₁₀ carbocyclyl or 5-10 memberedheterocyclyl of the formula (xiii):

wherein:

X is N, —NR³⁰, O, S or CR³¹R³²;

p is 0, 1 or 2;

each instance of R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹, R³¹ andR³² is independently selected from hydrogen, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OR^(B1), —ON(R^(B2))₂, —N(R^(B2))₂,—N(OR^(B3))R^(B3), —SH, —SR^(B1), —SSR^(B3), —C(═O)R^(B1), —CO₂H, —CHO,—C(OR^(B3))₂, —CO₂R^(B1), —OC(═O)R^(B1), —OCO₂R^(B1), —C(═O)N(R^(B2))₂,—OC(═O)N(R^(B2))₂, —NR^(B2)C(═O)R^(B1), —NR^(B2)CO₂R^(B1),—NR^(B2)C(═O)N(R^(B2))₂, —C(═NR^(B2))OR^(B1), —OC(═NR^(B2))R^(B1),—OC(═NR^(B2))OR^(B1), —C(═NR^(B2))N(R^(B2))₂, —OC(═NR^(B2))N(R^(B2))₂,—NR^(B2)C(═NR^(B2))N(R^(B2))₂, —C(═O)NR^(B2)SO₂R^(B1),—NR^(B2)SO₂R^(B1), —SO₂N(R^(B2))₂, —SO₂R^(B1), —SO₂OR^(B1), —OSO₂R^(B1),—S(═O)R^(B1), —OS(═O)R^(B1), —Si(R^(B1))₃,—OSi(R^(B1))₃—C(═S)N(R^(B2))₂, —C(═O)SR^(B1), —C(═S)SR^(B1),—SC(═S)SR^(B1), —P(═O)₂R^(B1), —OP(═O)₂R^(B1), —P(═O)(R^(B1))₂,—OP(═O)(R^(B1))₂, —OP(═O)(OR^(B3))₂, —P(═O)₂N(R^(B2))₂,—OP(═O)₂N(R^(B2))₂, —P(═O)(NR^(B2))₂, —OP(═O)(NR^(B2))₂,—NR^(B2)P(═O)(OR^(B3))₂, —NR^(B2)P(═O)(NR^(B2))₂, —P(R^(B3))₂,—P(R^(B3))₃, —OP(R^(B3))₂, —OP(R^(B3))₃, —B(OR^(B3))₂, or—BR^(B1)(OR^(B3)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, 5-14 membered heteroaryl, -L-R^(D) and —R^(E);or one or more of R²⁹ and R²¹, R²² and R³¹, R³² and R²³, R²⁴ and R²⁵,R²⁶ and R²⁷, R²⁸ and R²⁹, and R²⁶ and R²⁹, are joined to form a doublebond or a C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl or5-14 membered heteroaryl ring; optionally wherein X is N, then N and R²¹or N and R²³ are joined to form a double bond;

R³⁰ is selected from hydrogen, —OH, —OR^(B1), —N(R^(B3))₂, —CN,—C(═O)R^(B1), —C(═O)N(R^(B3))₂, —CO₂R^(B1), —SO₂R^(B1),—C(═NR^(B3))OR^(B1), —C(═NR^(B3))N(R^(B3))₂, —SO₂N(R^(B3))₂, —SO₂R^(B3),—SO₂OR^(B3), —S(═O)R^(B1), —C(═S)N(R^(B3))₂, —C(═O)SR^(B3),—C(═S)SR^(B3), —P(═O)₂R^(B1), —P(═O)(R^(B1))₂, —P(═O)₂N(R^(B3))₂,—P(═O)(NR^(B3))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or R²² and R³⁰or R³⁰ and R²³ are joined to form a 3-14 membered heterocyclyl or 5-14membered heteroaryl ring;

wherein:

each instance of R^(B1) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl,3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl;

each instance of R^(B2) is, independently, selected from hydrogen, —OH,—OR^(B1), —N(R^(B3))₂, —CN, —C(═O)R^(B1), —C(═O)N(R_(B3))₂, —CO₂R^(B1),—SO₂R^(B1), —C(═NR^(B3))OR^(B1), —C(═NR^(B3))N(R^(B3))₂, —SO₂N(R^(B3))₂,—SO₂R^(B3), —SO₂OR^(B3), —S(═O)R^(B1), —C(═S)N(R^(B3))₂, —C(═O)SR^(B3),—C(═S)SR^(B3), —P(═O)₂R^(B1), —P(═O)(R^(B1))₂, —P(═O)₂N(R^(B3))₂,—P(═O)(NR^(B3))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B2)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring;

each instance of R^(B3) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, or two R^(B3) groups are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring;

and L, R^(D) and R^(E) are as defined above and herein.

In certain embodiments, at least one of R²¹, R²², R²³, R²⁴, R²⁵, R²⁶,R²⁷, R²⁸, R²⁹, R³⁰, R³¹, and R³² is the group -L-R^(D) as defined aboveand herein. In certain embodiments, at least one of at least one of R²¹,R²², R²³, R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹, R³⁰, R³¹, and R³² is selectedfrom —R^(E) as defined herein.

In certain embodiments, p is 0. In certain embodiments, p is 1. Incertain embodiments, p is 2.

In certain embodiments, X is N. In certain embodiments, X is NR³⁰. Incertain embodiments, X is O. In certain embodiments, X is S. In certainembodiments, X is CR³¹R³²

For example, in certain embodiments, X is O. In certain embodiments,R^(B) is a 5-10 membered heterocyclyl of the formulae (xii-a) or(xiii-a):

wherein p, R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹ and R³⁰ are asdefined above and herein.

In certain embodiments, X is CR³¹R³². For example, in certainembodiments, R^(B) heterocyclyl of the formulae (xii-b) or (xiii-b);

is C₅₋₁₀ carbocyclyl of the formula (xii-c):

wherein p, R²¹, R²², R²³, R²⁴, R²⁵, R²⁶, R²⁷, R²⁸, R²⁹, R³¹ and R³² areas defined above and herein.

Joined Groups R^(B) and R^(C)

As described generally above, in certain embodiments, R^(B) and R^(C)together with the nitrogen (N) atom to which each is attached are joinedto form a 5-14 membered ring.

For example, in certain embodiments, R^(B) and R^(C) together with thenitrogen (N) atom to which each is attached are joined to form a 5-14membered ring of the formula (xiv):

wherein:

Q is N, —NR⁴⁰, O, S, or CR⁴¹R⁴²

m is 0, 1 or 2; and

each instance of R⁴¹, R⁴², R⁴³, R⁴⁴, R⁴⁵, R⁴⁶, R⁴⁷, R⁴⁸, R⁴⁹ and R⁵⁰ isindependently selected from hydrogen, halogen, —CN, —NO₂, —N₃, —SO₂H,—SO₃H, —OH, —OR^(F1), —ON(R^(F2))₂, —N(R^(F2))₂, —N(OR^(F3))R^(F3), —SH,—SR^(F1), —SSR^(F3), —C(═O)R^(F1), —CO₂H, —CHO, —C(OR^(F3))₂,—CO₂R^(F1), OC(═O)R^(F1), —OCO₂R^(F1), —C(═O)N(R^(F2))₂,—OC(═O)N(R^(F2))₂, —NR^(F2)C(═O)R^(F1), —NR^(F2)CO₂RF,—NR^(F2)C(═O)N(R^(F2))₂, —C(═NR^(F2))OR^(F1), —OC(═NR^(F2))R^(F1),—OC(═NR^(F2))OR^(F1), —C(═NR^(F2))N(R^(F2))₂, —OC(═NR^(F2))N(R^(F2))₂,—NR^(F2)C(═NR^(F2))N(R^(F2))₂, —C(═O)NR^(F2)SO₂R^(BC1), —NR^(F2)SO₂RF,—SO₂N(R^(F2))₂, —SO₂R^(F1), —SO₂OR^(F1), —OSO₂R^(F1), —S(═O)R^(F1),—OS(═O)R^(F1), —Si(R^(F1))₃, —OSi(R^(F1))₃—C(═S)N(R^(F2))₂,—C(═O)SR^(F1), —C(═S)SR^(F1), —SC(═S)SR^(F1), —P(═O)₂R^(F1),—OP(═O)₂R^(F1), —P(═O)(R^(F1))₂, —OP(═O)(R^(F1))₂, —OP(═O)(OR^(F3))₂,—P(═O)₂N(R^(F2))₂, —OP(═O)₂N(R^(F2))₂, —P(═O)(NR^(F2))₂,—OP(═O)(NR^(F2))₂, —NR^(F2)P(═O)(OR^(F3))₂, —NR^(F2)P(═O)(NR^(F2))₂,—P(R^(F3))₂, —P(R^(F3))₃, —OP(R^(F3))₂, —OP(R^(F3))₃, —B(OR^(F3))₂, or—BR^(F1)(OR^(F3)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, 5-14 membered heteroaryl, -L-R^(D) and —R^(E);or one or more of R⁴⁷ and R⁴⁹, R⁴⁸ and R⁵⁰, R⁴⁹ and R⁴¹, R⁵⁰ and R⁴²,R⁴¹ and R⁴⁵, R⁴² and R⁴⁶, R⁴⁵ and R⁴³, and R⁴⁶ and R⁴⁴, are joined toform a double bond or a C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl or 5-14 membered heteroaryl ring; optionally wherein Q is N,then N and R⁴⁹ or N and R⁴⁶ are joined to form a double bond;

R⁴⁰ is selected from hydrogen, —OH, —OR^(F1), —N(R^(F3))₂, —CN,—C(═O)R^(F1), —C(═O)N(R^(F3))₂, —CO₂R^(F1), —SO₂R^(F1),—C(═NR^(F3))OR^(F1), —C(═NR^(F3))N(R^(F3))₂, —SO₂N(R^(F3))₂, —SO₂R^(F3),—SO₂OR^(F3), —SOR^(F1), —C(═S)N(R^(F3))₂, —C(═O)SR^(F3), —C(═S)SR^(F3),—P(═O)₂R^(F1), —P(═O)(R^(F1))₂, —P(═O)₂N(R^(F3))₂, —P(═O)(NR^(F3))₂,—C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, optionally wherein R⁴⁹ and R⁴⁰or R⁴⁰ and R⁴⁵ are joined to form a 3-14 membered heterocyclyl, or 5-14membered heteroaryl ring;

wherein:

each instance of R^(F1) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl,3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl;

each instance of R^(F2) is, independently, selected from hydrogen, —OH,—OR^(F1), —N(R^(F3))₂, —CN, —C(═O)R^(F1), —C(═O)N(R^(F3))₂, —CO₂R^(F1),—SO₂R^(F1), —C(═NR^(F3))OR^(F1), —C(═NR^(F3))N(R^(F3))₂, —SO₂N(R^(F3))₂,—SO₂R^(F3), —SO₂OR^(F3), —S(═O)R^(F1), —C(═S)N(R^(F3))₂, —C(═O)SR^(F3),—C(═S)SR^(F3), —P(═O)₂R^(F1), —P(═O)(R^(F1))₂, —P(═O)₂N(R^(F3))₂,—P(═O)(NR^(F3))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(F2)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring;

each instance of R^(F3) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, or two RF³ groups are joined to forma 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring;

and L, R^(D) and R^(E) are as defined above and herein.

In certain embodiments, at least one of R⁴⁰, R⁴¹, R⁴², R⁴³, R⁴⁴, R⁴⁵,R⁴⁶, R⁴⁷, R⁴⁸, R⁴⁹ and R⁵⁰ is the group -L-R^(D) as defined above andherein. In certain embodiments, at least one of R⁴⁰, R⁴¹, R⁴², R⁴³, R⁴⁴,R⁴⁵, R⁴⁶, R⁴⁷, R⁴⁸, R⁴⁹ and R⁵⁰ is selected from —R^(E) as definedherein.

In certain embodiments, m is 0. In certain embodiments, m is 1. Incertain embodiments, m is 2.

In certain embodiments, Q is N. In certain embodiments, Q is NR⁴⁰. Incertain embodiments, Q is O. In certain embodiments, Q is S. In certainembodiments, Q is CR⁴¹R⁴²

In certain embodiments, R⁴⁷ and R⁴⁹ are joined to form a double bond andR⁴⁸ and R⁵⁰ are joined to form a C₆₋₁₄ aryl or 5-14 membered heteroaryl.For example, in certain embodiments, R^(B) and R^(C) together with thenitrogen (N) atom to which each is attached are joined to form a 5-14membered ring of the formula (xv):

wherein Q, m, W, R⁴¹, R⁴², R⁴³, R⁴⁴, R⁴⁵, R⁴⁶, R⁶, R⁷, R⁸ and R⁹ are asdefined above and herein.

In certain embodiments, Q is CR⁴¹R⁴², R⁴⁹ and R⁴¹ are joined to form adouble bond and R⁵⁰ and R⁴² are joined to form a C₆₋₁₄ aryl or 5-14membered heteroaryl. For example, in certain embodiments, R^(B) andR^(C) together with the nitrogen (N) atom to which each is attached arejoined to form a group of the formula (xvi):

wherein m, W, R⁴³, R⁴⁴, R⁴⁵, R⁴⁶, R⁴⁷ and R⁴⁸ are as defined above andherein; and

wherein R⁶⁶, R⁶⁷, R⁶⁸ and R⁶⁹ are independently selected from the groupconsisting of hydrogen, halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH,—OR^(F4), —ON(R^(F5))₂, —N(R^(F5))₂, —N(OR^(F6))R^(F6), —SH, —SR^(F4),—SSR^(F6), —C(═O)R^(F4), —CO₂H, —CHO, —C(OR^(F6))₂, —CO₂R^(F4),—OC(═O)R^(F4), —OCO₂R^(F4), —C(═O)N(R^(F5))₂, —OC(═O)N(R^(F5))₂,—NR^(F5)C(═O)R^(F4), —NR^(F5)CO₂R^(F4), —NR^(F5)C(═O)N(R^(F5))₂,—C(═NR^(F5))OR^(F4), —OC(═NR^(F5))R^(F4), —OC(═NR^(F5))OR^(F4),—C(═NR^(F5))N(R^(F5))₂, —OC(═NR^(F5))N(R^(F5))₂,—NR^(F5)C(═NR^(F5))N(R^(F5))₂, —C(═O)NR^(F5)SO₂R^(F4),—NR^(F5)SO₂R^(F4), —SO₂N(R^(F5))₂, —SO₂R^(F4), —SO₂OR^(F4), —OSO₂R^(F4),—S(═O)R^(F4), —OS(═O)R^(F4), —Si(R^(F4))₃,—OSi(R^(F4))₃—C(═S)N(R^(F5))₂, —C(═O)SR^(F4), —C(═S)SR^(F4),—SC(S)SR^(F4), —P(═O)₂R^(F4), —OP(═O)₂R^(F4), —P(═O)(R^(F4))₂,—OP(═O)(R^(F4))₂, —OP(═O)(OR^(F6))₂, —P(═O)₂N(R^(F5))₂,—OP(═O)₂N(R^(F5))₂, —P(═O)(NR^(F5))₂, (═O)₂, —NR^(F5)P(═O)(OR^(F6))₂,—NR^(F5)P(═O)(NR^(F5))₂, —P(R^(F6))₂, —P(R^(F6))₃, —OP(R^(F6))₂,—OP(R^(F6))₃, —B(OR^(F6))₂, or —BR^(F4)(OR^(F6)), C₁₋₁₀ alkyl, C₁₋₁₀perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, 5-14 membered heteroaryl, -L-R^(D) and —R^(E); or one or more ofR⁶⁶ and R⁶⁷, R⁶⁷ and R⁶⁸, and R⁶⁸ and R⁶⁹ are joined to form a C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl or 5-14 memberedheteroaryl ring;

each instance of R^(F4) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl;

each instance of R^(F5) is, independently, selected from hydrogen, —OH,—OR^(F4), —N(R^(F6))₂, —CN, —C(═O)R^(F4), —C(═O)N(R^(F6))₂, —CO₂R^(F4),—SO₂R^(F4), —C(═NR^(F6))OR^(F4), —C(═NR^(F6))N(R^(F6))₂, —SO₂N(R^(F6))₂,—SO₂R^(F6), —SO₂OR^(F6), —SOR^(F4), —C(═S)N(R^(F6))₂, —C(═O)SR^(F6),—C(═S)SR^(F6), —P(═O)₂R^(F4), —P(═O)(R^(F4))₂, —P(═O)₂N(R^(F6))₂,—P(═O)(NR^(F6))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(F5)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring; and

each instance of R^(F6) is, independently, selected from hydrogen, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, or two R^(F6) groups are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring.

In certain embodiments, at least one of R⁴³, R⁴⁴, R⁴⁵, R⁴⁶, R⁴⁷, R⁴⁸,R⁶⁶, R⁶⁷, R⁶⁸ and R⁶⁹ is the group -L-R^(D) as defined above and herein.In certain embodiments, at least one of R⁴³, R⁴⁴, R⁴⁵, R⁴⁶, R⁴⁷, R⁴⁸,R⁶⁶, R⁶⁷, R⁶⁸ and R⁶⁹ is selected from —R^(E) as defined herein.

In certain embodiments, m is 0. In certain embodiments, m is 1. Incertain embodiments, m is 2.

Group R^(C)

As described generally above, R^(C) is selected from hydrogen, —OH,—OR^(C1), —ON(R^(C2))₂, —N(R^(C2))₂, —C(═O)R^(C1), —CHO, —CO₂R^(C1),—C(═O)N(R^(C2))₂, —C(═NR^(C2))OR^(C1), —C(═NR^(C2))N(R^(C2))₂,—SO₂R^(C1), —S(═O)R^(C1), —Si(R^(C1))₃, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl,C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl;

wherein:

each instance of R^(C1) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl,3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl;and

each instance of R^(C2) is, independently, selected from hydrogen, —OH,—OR^(C1), —N(R^(C3))₂, —CN, —C(═O)R^(C1), —C(═O)N(R^(C3))₂, —CO₂R^(C1),—SO₂R^(C1), —C(═NR^(C3))OR^(C1), —C(═NR^(C3))N(R^(C3))₂, —SO₂N(R^(C3))₂,—SO₂R^(C3), —SO₂OR^(C3), —SOR^(C1), —C(═S)N(R^(C3))₂, —C(═O)SR^(C3),—C(═S)SR^(C3), —P(═O)₂R^(C1), —P(═O)(R¹)₂, —P(═O)₂N(R^(C3))₂,—P(═O)(NR^(C3))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(C2)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring;

or R^(B) and R^(C) together with the nitrogen (N) atom to which each isattached are joined to form a 5-14 membered ring.

In certain embodiments, R^(C) is selected from C₁₋₁₀ alkyl, C₁₋₁₀perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl.

In certain embodiments, R^(C) is an unsubstituted group, e.g., selectedfrom unsubstituted C₁₋₁₀ alkyl, unsubstituted C₂₋₁₀ alkenyl,unsubstituted C₂₋₁₀ alkynyl, unsubstituted 3-14 memberedheteroaliphatic, unsubstituted C₃₋₁₀ carbocyclyl, unsubstituted 3-14membered heterocyclyl, unsubstituted C₆₋₁₄ aryl and unsubstituted 5-14membered heteroaryl. However, in certain embodiments, R^(C) is anunsubstituted group wherein —CH₃ and —CH₂CH₃ are excluded.

In certain embodiments, R^(C) is a group having 2 or more carbon atoms,e.g., selected from C₂₋₁₀ alkyl, C₂₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl,C₂₋₁₀ alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl. Incertain embodiments, R^(C) is an unsubstituted group having 2 or morecarbon atoms. However, in certain embodiments, R^(C) is a group having 2or more carbon atoms wherein —CH₂CH₃ is excluded.

In certain embodiments, R^(C) is a group having 3 or more carbon atoms,e.g., selected from C₃₋₁₀ alkyl, C₃₋₁₀ perhaloalkyl, C₃₋₁₀ alkenyl,C₃₋₁₀ alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl. Incertain embodiments, R^(C) is an unsubstituted group having 3 or morecarbon atoms. However, in certain embodiments, R^(C) is a group having 3or more carbon atoms wherein —CH(CH₃)₂ is excluded.

In certain embodiments, R^(C) is a group having 4 or more carbon atoms,e.g., selected from C₄₋₁₀ alkyl, C₄₋₁₀ perhaloalkyl, C₄₋₁₀ alkenyl,C₄₋₁₀ alkynyl, 5-14 membered heteroaliphatic, C₅₋₁₀ carbocyclyl, 5-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl. Incertain embodiments, R^(C) is an unsubstituted group having 4 or morecarbon atoms.

In certain embodiments, R^(C) is an acyclic group, e.g., selected fromC₁₋₁₀ alkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl and 3-14 memberedheteroaliphatic. In certain embodiments, R^(C) is an unsubstitutedacyclic group, e.g., selected from unsubstituted C₁₋₁₀ alkyl,unsubstituted C₂₋₁₀ alkenyl, unsubstituted C₂₋₁₀ alkynyl andunsubstituted 3-14 membered heteroaliphatic. However, in certainembodiments, R^(C) is an acyclic group, wherein —CH₃ and —CH₂CH₃ areexcluded.

In certain embodiments, R^(C) is C₁₋₁₀ alkyl. In certain embodiments,R^(C) is an unsubstituted C₁₋₁₀ alkyl. In certain embodiments, R^(C) isC₁₋₁₀ alkyl, wherein —CH₃ is excluded. In certain embodiments, R^(C) isC₁₋₁₀ alkyl, wherein —CH₂CH₃ is excluded. In certain embodiments, R^(C)is C₁₋₁₀ alkyl, wherein —CH(CH₃)₂ is excluded.

In certain embodiments, R^(C) is C₂₋₁₀ alkyl, e.g., selected from ethyl,n-propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, iso-butyl,n-pentyl, pentan-3-yl, amyl, neopentyl, 3-methyl-2-butanyl, tertiaryamyl and n-hexyl. In certain embodiments, R^(C) is an unsubstitutedC₂₋₁₀ alkyl. In certain embodiments, R^(C) is C₂₋₁₀ alkyl, wherein—CH₂CH₃ is excluded. In certain embodiments, R^(C) is C₂₋₁₀ alkyl,wherein —CH(CH₃)₂ is excluded.

In certain embodiments, R^(C) is C₃₋₁₀ alkyl, e.g., selected fromn-propyl, isopropyl, n-butyl, tert-butyl, sec-butyl, iso-butyl,n-pentyl, pentan-3-yl, amyl, neopentyl, 3-methyl-2-butanyl, tertiaryamyl and n-hexyl. In certain embodiments, R^(C) is an unsubstitutedC₃₋₁₀ alkyl. In certain embodiments, R^(C) is C₃₋₁₀ alkyl, wherein—CH(CH₃)₂ is excluded.

In certain embodiments, R^(C) is C₄₋₁₀ alkyl, e.g., selected fromn-butyl, tert-butyl, sec-butyl, iso-butyl, n-pentyl, pentan-3-yl, amyl,neopentyl, 3-methyl-2-butanyl, tertiary amyl and n-hexyl. In certainembodiments, R^(C) is an unsubstituted C₄₋₁₀ alkyl.

In certain embodiments, R^(C) is C₂₋₁₀ alkenyl. In certain embodiments,R^(C) is an unsubstituted C₂₋₁₀ alkenyl. In certain embodiments, R^(C)is C₂₋₁₀ alkenyl selected from allyl.

In certain embodiments, R^(C) is C₂₋₁₀ alkynyl. In certain embodiments,R^(C) is an unsubstituted C₂₋₁₀ alkynyl.

In certain embodiments, R^(C) is 3-14 membered heteroaliphatic. Incertain embodiments, R^(C) is an unsubstituted 3-14 memberedheteroaliphatic.

In certain embodiments, R^(C) is a cyclic group, e.g., selected fromC₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl and 5-14membered heteroaryl. In certain embodiments, R^(C) is an unsubstitutedcyclic group, e.g., selected from unsubstituted C₃₋₁₀ carbocyclyl,unsubstituted 3-14 membered heterocyclyl, unsubstituted C₆₋₁₄ aryl andunsubstituted 5-14 membered heteroaryl.

In certain embodiments, R^(C) is C₃₋₁₀ carbocyclyl. In certainembodiments, R^(C) is C₄₋₁₀ carbocyclyl. In certain embodiments, R^(C)is C₅₋₁₀ carbocyclyl. In certain embodiments, R^(C) is C₅₋₈ carbocyclyl.In certain embodiments, R^(C) is C₃₋₁₀ carbocyclyl selected fromcyclopropyl (C₃), cyclobutyl (C₄), cyclopentyl (C₅), cyclopentenyl (C₅),cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), cycloheptyl(C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇) and cyclooctyl (C₈).In certain embodiments, R^(C) is C₃₋₁₀ carbocyclyl selected fromcyclopentyl and cyclohexyl. In certain embodiments, R^(C) is anunsubstituted C₃₋₁₀ carbocyclyl.

In certain embodiments, R^(C) is 3-14 membered heterocyclyl. In certainembodiments, R^(C) is 5-10 membered heterocyclyl. In certainembodiments, R^(C) is 5-6 membered heterocyclyl. In certain embodiments,R^(C) is 3-14 membered heterocyclyl selected from azirdinyl, oxiranyl,thiorenyl, azetidinyl, oxetanyl, thietanyl, tetrahydrofuranyl,dihydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl,dihydropyrrolyl, dioxolanyl, oxathiolanyl, dithiolanyl, piperidinyl,tetrahydropyranyl, dihydropyridinyl, thianyl, piperazinyl, morpholinyl,dithianyl, dioxanyl, azepanyl, oxepanyl thiepanyl, azocanyl, oxecanyland thiocanyl. In certain embodiments, R^(C) is 3-14 memberedheterocyclyl selected from tetrahydropyranyl. In certain embodiments,R^(C) is an unsubstituted 3-14 membered heterocyclyl.

In certain embodiments, R^(C) is C₆₋₁₄ aryl. In certain embodiments,R^(C) is a C₆₋₁₄ aryl selected from phenyl, naphthyl and anthracyl. Incertain embodiments, R^(C) a C₆₋₁₄ aryl selected from phenyl. In certainembodiments, R^(C) is an unsubstituted C₆₋₁₄ aryl.

In certain embodiments, R^(C) is 5-14 membered heteroaryl. In certainembodiments, R^(C) is 5-10 membered heteroaryl. In certain embodiments,R^(C) is 5-6 membered heteroaryl. In certain embodiments, R^(C) is a5-membered heteroaryl, e.g., selected from pyrrolyl, furanyl,thiophenyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl,isothiazolyl, triazolyl, oxadiazolyl, thiadiazolyl and tetrazolyl. Incertain embodiments, R^(A) is a 6-membered heteroaryl, e.g., selectedfrom pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl andtetrazinyl. In certain embodiments, R^(C) is an unsubstituted 5-14membered heteroaryl.

Exemplary Combinations of Groups R^(A), R^(B) and R^(C)

Various combinations of R^(A), R^(B) and/or R^(C) are contemplatedherein, and are described in more detail below and herein.

For example, in certain embodiments, both R^(B) and R^(C) are cyclic,i.e., R^(B) is selected from C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl and 5-14 membered heteroaryl, and R^(C) isselected from C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl,and 5-14 membered heteroaryl. In certain embodiments, R^(C) is a grouphaving 2 or more carbon atoms. In certain embodiments, R^(C) is a grouphaving 3 or more carbon atoms. In certain embodiments, R^(C) is a grouphaving 4 or more carbon atoms. In certain embodiments, R^(C) is anunsubstituted cyclic group.

In certain embodiments, R^(B) is cyclic and R^(C) is acyclic, i.e.,R^(B) is selected from C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl and 5-14 membered heteroaryl, and R^(C) is selected from —OH,—OR^(C1), —ON(R^(C2))₂, —N(R^(C2))₂, —C(═O)R^(C1), —CHO, —CO₂R^(C1),—C(═O)N(R^(C2))₂, —C(═NR^(C2))OR^(C1), —C(═NR^(C2))N(R^(C2))₂,—SO₂R^(C1), —S(═O)R^(C1), —Si(R^(C1))₃, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl,C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 membered heteroaliphatic, whereR^(C1) and R^(C2) are as defined above and herein. In certainembodiments, R^(C) is an acyclic group having 2 or more carbon atoms. Incertain embodiments, R^(C) is an acyclic group having 3 or more carbonatoms. In certain embodiments, R^(C) is an acyclic group having 4 ormore carbon atoms. In certain embodiments, R^(C) is an unsubstitutedacyclic group.

In certain embodiments, R^(A) and R^(B) are independently selected fromC₆₋₁₄ aryl and 5-14 membered heteroaryl. In certain embodiments, R^(A)is C₆₋₁₄ aryl, and R^(B) is C₆₋₁₄ aryl or 5-14 membered heteroaryl. Incertain embodiments, R^(A) is 5-14 membered heteroaryl, and R^(B) isC₆₋₁₄ aryl or 5-14 membered heteroaryl. In certain embodiments, R^(A) isC₆₋₁₄ aryl or 5-14 membered heteroaryl, and R^(B) is C₆₋₁₄ aryl. Incertain embodiments, R^(A) is C₆₋₁₄ aryl or 5-14 membered heteroaryl,and R^(B) is 5-14 membered heteroaryl.

In certain embodiments, both R^(A) and R^(B) are C₆₋₁₄ aryl. In certainembodiments, both R^(A) and R^(B) are phenyl (C₆ aryl). In certainembodiments, R^(A) is C₆₋₁₄ aryl and R^(B) is C₃₋₁₀ carbocyclyl.

In certain embodiments, R^(A) is C₆₋₁₄ aryl and R^(B) is 5-14 memberedheteroaryl.

In certain embodiments, R^(A) is C₆₋₁₄ aryl and R^(B) is 3-14 memberedheterocyclyl.

In certain embodiments, R^(A) is C₆₋₁₄ aryl and R^(B) and R^(C) togetherwith the nitrogen (N) atom to which each is attached are joined to forma 5-14 membered ring.

In certain embodiments, both R^(A) and R^(B) are 5-14 memberedheteroaryl.

In certain embodiments, R^(A) is 5-14 membered heteroaryl and R^(B) isC₃₋₁₀ carbocyclyl.

In certain embodiments, R^(A) is 5-14 membered heteroaryl and R^(B) isC₆₋₁₄ aryl.

In certain embodiments, R^(A) is 5-14 membered heteroaryl and R^(B) is3-14 membered heterocyclyl.

In certain embodiments, R^(A) is 5-14 membered heteroaryl, and R^(B) andR^(C) together with the nitrogen (N) atom to which each is attached arejoined to form a 5-14 membered ring.

In certain embodiments, the compound is of the formula (II):

or a pharmaceutically acceptable form thereof,

wherein R^(C), W—R¹, W—R², W—R³, W—R⁴, W—R⁵, W—R⁶, W—R⁷, W—R⁸, W—R⁹, andW—R¹ are as defined above and herein.

In certain embodiments, at least one of R⁶, R⁷, R⁸, R⁹ and R¹⁰ of theformula (II) is the group -L-R^(D) as defined above and herein. Incertain embodiments, at least one of R⁶, R⁷, R⁸, R⁹ and R¹⁰ of theformula (II) is further selected from the group —R^(E) as defined aboveand herein.

In certain embodiments, the compound is of the formulae (II-a), (II-b)or (II-c):

or a pharmaceutically acceptable form thereof;

wherein R^(C), W—R¹, W—R², W—R³, W—R⁴, W—R⁵, W—R⁷, W—R⁸, W—R⁹, and W—R¹⁰are as defined above and herein.

In certain embodiments, at least one of R⁷, R⁸, R⁹ and R¹⁰ of theformulae (II-a), (II-b) or (II-c) is the group -L-R^(D) as defined aboveand herein. In certain embodiments, at least one of R⁷, R⁸, R⁹ and R¹⁰of the formulae (II-a), (II-b) or (II-c) is further selected from thegroup —R^(E) as defined above and herein.

In certain embodiments, the compound is of the formula (III):

or a pharmaceutically acceptable form thereof,

wherein R^(C), R¹, R², R³, R⁴, R⁵, W—R⁶, W—R⁷, W—R, W—R⁹, and W—R¹⁰ areas defined above and herein.

In certain embodiments, at least one of R⁶, R⁷, R⁸, R⁹ and R¹⁰ of thecompound of formula (III) is the group -L-R^(D) as defined above andherein. In certain embodiments, at least one of R⁶, R⁷, R⁸, R⁹ and R¹⁰of the compound of formula (III) is further selected from the group—R^(E) as defined above and herein.

In certain embodiments, the compound is of the formulae (III-a), (III-b)or (III-c):

or a pharmaceutically acceptable form thereof,

wherein R^(C), R¹, R², R³, R⁴, R⁵, W—R⁷, W—R⁸, W—R⁹, and W—R¹⁰ are asdefined above and herein.

In certain embodiments, at least one of R⁷, R⁸, R⁹ and R¹⁰ of theformulae (III-a), (III-b) or (III-c) is the group -L-R^(D) as definedabove and herein. In certain embodiments, at least one of R⁷, R⁸, R⁹ andR¹⁰ of formulae (III-a), (III-b) or (III-c) is further selected from thegroup —R^(E) as defined above and herein.

In certain embodiments, the compound is of the formula (IV):

or a pharmaceutically acceptable form thereof;

wherein R^(C), R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are asdefined above and herein.

In certain embodiments, at least one of R⁶, R⁷, R⁸, R⁹ and R¹⁰ of theformula (IV) is the group -L-R^(D) as defined above and herein. Incertain embodiments, at least one of R⁶, R⁷, R⁸, R⁹ and R¹⁰ of theformula (IV) is further selected from the group —R^(E) as defined aboveand herein. In certain embodiments, R¹—R⁵ are independently H, C₁₋₁₀alkyl, C₁₋₁₀ alkyloxy, C₆₋₁₄ aryloxy, CN, —SO₂N(R^(A7))₂, —SO₂R^(A6),and —SO₂OR^(A6); R^(C) is unsubstituted C₁₋₁₀ alkyl or unsubstitutedC₃₋₁₀ carbocyclyl; and R⁶-R¹⁰ are independently selected from H, C₁₋₁₀alkyl, C₁₋₁₀ alkyloxy, C₆₋₁₄ aryloxy, COOH, and —CO₂R^(A6). In certainembodiments, R¹—R⁵ are independently H, methyl, methoxy, CN, and SO₂Me;R^(C) is unsubstituted C₁₋₃ alkyl or unsubstituted C₅₋₆ cycloalkyl; andR⁶-R¹⁰ are independently selected from H, methyl, methoxy, phenoxy,COOH, and CO₂Me.

In certain embodiments, the compound is of the formulae (IV-a), (IV-b),(IV-c) or (IV-d):

or a pharmaceutically acceptable form thereof,

wherein R^(C), R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are asdefined above and herein.

In certain embodiments, at least one of R⁷, R⁸, R⁹ and R¹⁰ of theformulae (IV-a), (IV-b) or (IV-c) is the group -L-R^(D) as defined aboveand herein. In certain embodiments, at least one of R⁷, R⁸, R⁹ and R¹⁰of the formulae (IV-a), (IV-b), (IV-c) or (IV-d) is further selectedfrom the group —R^(E) as defined above and herein.

In one embodiment, provided herein is a compound of formula (IV-d), or apharmaceutically acceptable form thereof. In one embodiment where thecompound is of formula (IV-d), R^(C) is C₁₋₁₀alkyl or C₃₋₁₀carbocyclyl.In one embodiment, R^(C) is ethyl, isopropyl, cyclopentyl or cyclohexyl.

In another embodiment where the compound is of formula (IV-d), R¹ and R²are each independently hydrogen, halogen, —CN, —OR^(A1) or —SO₂R^(A1),wherein R^(A1) is C₁₋₁₀ alkyl. In another embodiment, R¹ and R² are eachindependently hydrogen, fluoro, methoxy, —CN or —SO₂CH₃.

In another embodiment where the compound is of formula (IV-d), R⁶ and R⁷are each independently hydrogen, halogen or —O—R^(B1), wherein R^(B1) isC₁₋₁₀ alkyl or C₆₋₁₄ aryl. In another embodiment, R⁶ and R⁷ are eachindependently hydrogen, fluoro, methoxy or phenyloxy.

In certain embodiments, the compound is of the formula (V):

or a pharmaceutically acceptable form thereof,

wherein R^(C), X, Y, Z, R¹, R², R³, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are asdefined above and herein.

In certain embodiments, at least one of R⁶, R⁷, R⁸, R⁹ and R¹⁰ of theformula (V) is the group -L-R^(D) as defined above and herein. Incertain embodiments, at least one of R⁶, R⁷, R⁸, R⁹ and R¹⁰ of theformula (V) is further selected from the group —R^(E) as defined aboveand herein.

In certain embodiments, the compound is of the formulae (V-a), (V-b) or(V-c):

or a pharmaceutically acceptable form thereof,

wherein R^(C), X, Y, Z, R¹, R², R³, R⁷, R⁸, R⁹, and R¹⁰ are as definedabove and herein.

In certain embodiments, at least one of R⁷, R⁸, R⁹ and R¹⁰ of theformulae (V-a), (V-b) or (V-c) is the group -L-R^(D) as defined aboveand herein. In certain embodiments, at least one of R⁷, R⁸, R⁹ and R¹⁰of the formulae (V-a), (V-b) or (V-c) is further selected from the group—R^(E) as defined above and herein.

In certain embodiments, the compound is of the formula (VI):

or a pharmaceutically acceptable form thereof;

wherein R^(C), Y, R¹, R², R³, R⁶, R⁷, R⁸, R⁹, and R¹⁰ are as definedabove and herein.

In certain embodiments, at least one of R⁶, R⁷, R⁸, R⁹ and R¹⁰ of theformula (VI) is the group -L-R^(D) as defined above and herein. Incertain embodiments, at least one of R⁶, R⁷, R⁸, R⁹ and R¹⁰ of theformula (VI) is further selected from the group —R^(E) as defined aboveand herein. In certain embodiments, R¹—R³ are independently H, C₁₋₁₀alkyl, C₁₋₁₀ alkyloxy, C₆₋₁₄ aryloxy, CN, —SO₂N(R^(A7))₂, —SO₂R^(A6),and —SO₂OR^(A6); R^(C) is unsubstituted C₁₋₁₀ alkyl or unsubstitutedC₃₋₁₀ carbocyclyl; and R⁶-R¹⁰ are independently selected from H, C₁₋₁₀alkyl, C₁₋₁₀ alkyloxy, C₆₋₁₄ aryloxy, COOH, and —CO₂R^(A6). In certainembodiments, R¹—R³ are independently H, methyl, methoxy, and CN; R^(C)is unsubstituted C₅₋₆ cycloalkyl; and R⁶-R¹⁰ are independently selectedfrom H, methyl, methoxy, phenoxy, COOH, and CO₂Me.

In certain embodiments, the compound is of the formulae (VI-a), (VI-b)or (VI-c):

or a pharmaceutically acceptable form thereof,

wherein R^(C), Y, R¹, R², R³, R⁷, R⁸, R⁹, and R¹⁰ are as defined aboveand herein.

In certain embodiments, at least one of R⁷, R⁸, R⁹ and R¹⁰ of theformulae (VI-a), (VI-b) or (VI-c) is the group -L-R^(D) as defined aboveand herein. In certain embodiments, at least one of R⁷, R⁸, R⁹ and R¹⁰of the formulae (VI-a), (VI-b) or (VI-c) is further selected from thegroup —R^(E) as defined above and herein.

Exemplary Compounds

Exemplary compounds are set forth in the Exemplification and listed inTables 1, 2, 3 and 4 provided therein.

In certain embodiments, a compound of formula (I) is selected from anyof the compounds provided in Tables 1, 2, 3 or 4. In certainembodiments, a compound of formula (I) is selected from any of thecompounds provided in Table 1. In certain embodiments, a compound offormula (I) is selected from any of the compounds provided in Table 2.In certain embodiments, a compound of formula (I) is selected from anyof the compounds provided in Table 3. In certain embodiments, a compoundof formula (I) is selected from any of the compounds provided in Table4.

In certain embodiments, a compound of formula (I) is selected from anyof the compounds provided in Tables 1, 2 or 3. In certain embodiments, acompound of formula (I) is selected from any of the compounds providedin Tables 1 or 2. In certain embodiments, a compound of formula (I) isselected from any of the compounds provided in Tables 1 or 3. In certainembodiments, a compound of formula (I) is selected from any of thecompounds provided in Tables 1 or 4. In certain embodiments, a compoundof formula (I) is selected from any of the compounds provided in Tables2 or 3. In certain embodiments, a compound of formula (I) is selectedfrom any of the compounds provided in Tables 2 or 4.

Activities provided from the FASN NADPH Consumption Assay are designatedin Tables 1, 2, 3 and 4 with a star (*), wherein “A*” refers tocompounds having an IC₅₀ of less than 60 nM; “B*” refers to compoundshaving an IC₅₀ of 60 nM to 250 nM, inclusive; “C*” refers to compoundshaving an IC₅₀ of greater than 250 nM to 1000 nM, inclusive; “D*” refersto compounds having an IC₅₀ of greater than 1000 nM to 10,000 nM,inclusive; and “E*” refers to compounds having an IC₅₀ of greater than10,000 nM, as measured by the assay.

Activities provided from the FASN Scintillation Proximity FlashplateAssay are provided in Tables 1, 2, 3 and 4, wherein “A” refers tocompounds having an IC₅₀ of less than 15 nM/mL; “B” refers to compoundshaving an IC₅₀ of 15 nM to 100 nM, inclusive; “C” refers to compoundshaving an IC₅₀ of greater than 100 nM to 200 nM, inclusive; “D” refersto compounds having an IC₅₀ of greater than 200 nM to 5000 nM,inclusive; and “E” refers to compounds having an IC₅₀ of greater than5000 nM, as measured by the assay.

In certain embodiments, a compound of formula (I) is any of thecompounds provided in Tables 1, 2 or 3 having an activity of “A”, “A*”,“B”, “B*”, “C” or “C*”. In certain embodiments, a compound of formula(I) is any of the compounds provided in Tables 1, 2 or 3 having anactivity of “A” or “A*”. In certain embodiments, a compound of formula(I) is any of the compounds provided in Tables 1, 2 or 3 having anactivity of “B” or “B*”. In certain embodiments, a compound of formula(I) is any of the compounds provided in Tables 1, 2 or 3 having anactivity of “C” or “C*”

In certain embodiments, compounds provided herein include any of thecompounds provided in Tables 1, 2 or 3 substituted with a group-L-R^(D), as defined above and herein, and having an activity of “A”,“A*”, “B” or “B*”

For example, in certain embodiments, the compound of formula (I) is acompound selected from the group consisting of:

or a pharmaceutically acceptable form thereof.

In certain embodiments, the compound of formula (I) is a compoundselected from the group consisting of:

or a pharmaceutically acceptable form thereof.

In other embodiments, the compound of formula (I) is a compound selectedfrom the group consisting of:

or a pharmaceutically acceptable form thereof.

3. Pharmaceutical Compositions and Formulations

In certain embodiments, provided herein is a pharmaceutical compositioncomprising at least one compound of formula (I) or a pharmaceuticallyacceptable form thereof, and one or more pharmaceutically acceptableexcipients.

In some embodiments, provided herein is a pharmaceutical compositioncomprising at least one compound of formula (I) or a pharmaceuticallyacceptable form thereof, as provided in Tables 1, 2, 3, or 4 and one ormore pharmaceutically acceptable excipients. In other embodiments,provided herein is a pharmaceutical composition comprising at least onecompound of formula (I) or a pharmaceutically acceptable form thereof,as provided in Tables 1, 2, or 3 having an activity of “A”, “A*”, “B” or“B*” and one or more pharmaceutically acceptable excipients. In otherembodiments, provided herein is a pharmaceutical composition comprisingat least one compound of formula (I) or a pharmaceutically acceptableform thereof, as provided in Tables 1, 2 or 3 having an activity of “A”or “A*” and one or more pharmaceutically acceptable excipients.

As described above, the pharmaceutical compositions provided herein cancomprise a “pharmaceutically acceptable excipient”, which, as usedherein, includes any and all solvents, diluents, or other liquidvehicle, dispersion or suspension aids, surface active agents, isotonicagents, thickening or emulsifying agents, preservatives, solid binders,lubricants and the like, as suited to the particular dosage formdesired. Remington's Pharmaceutical Sciences, 16th Ed., E. W. Martin(Mack Publishing Co., Easton, Pa., 1980) discloses variouspharmaceutically acceptable excipients used in formulatingpharmaceutically acceptable compositions and known techniques for thepreparation thereof. Except insofar as any conventional excipient mediumis incompatible with the compounds provided herein, such as by producingany undesirable biological effect or otherwise interacting in adeleterious manner with any other component(s) of the pharmaceuticallyacceptable composition, the excipient's use is contemplated to be withinthe scope of this disclosure. Some examples of materials which can serveas pharmaceutically acceptable excipients include, but are not limitedto, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, or potassium sorbate, partialglyceride mixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates,waxes, polyethylene-polyoxypropylene-block polymers, wool fat, sugarssuch as lactose, glucose and sucrose; starches such as corn starch andpotato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols; such apropylene glycol or polyethylene glycol; esters such as ethyl oleate andethyl laurate; agar; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol, and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in thepharmaceutically acceptable composition, according to the judgment ofthe formulator.

In some embodiments, a compound of formula (I) is administered at about0.01 mg/kg to about 200 mg/kg, such as at about 0.1 mg/kg to about 100mg/kg, further such as at about 0.5 mg/kg to about 50 mg/kg.

A “subject” to which administration is contemplated includes, but is notlimited to, humans (i.e., a male or female of any age group, e.g., apediatric subject (e.g., infant, child, adolescent) or adult subject(e.g., young adult, middle-aged adult or senior adult)) and/or otherprimates (e.g., cynomolgus monkeys, rhesus monkeys); mammals, includingcommercially relevant mammals such as cattle, pigs, horses, sheep,goats, cats, and/or dogs; and/or birds, including commercially relevantbirds such as chickens, ducks, geese, and/or turkeys.

The formulations of the pharmaceutically acceptable compositionsdescribed herein can be prepared by any method known or hereafterdeveloped in the art of pharmacology. In general, such preparatorymethods include the step of bringing the compound of formula (I) intoassociation with one or more pharmaceutically acceptable excipients andthen, if necessary and/or desirable, shaping and/or packaging theproduct into a desired single- or multi-dose unit.

A pharmaceutical composition provided herein can be prepared, packaged,and/or sold in bulk, as a single unit dose, and/or as a plurality ofsingle unit doses. As used herein, a “unit dose” is a discrete amount ofthe pharmaceutical composition comprising a predetermined amount of atleast one compound of formula (I). The amount of the compound of formula(I) is generally equal to the dosage of the compound of formula (I)which would be administered to a subject and/or a convenient fraction ofsuch a dosage such as, for example, one-half or one-third of such adosage.

The relative amounts of the compound of formula (I), thepharmaceutically acceptable excipient, and/or any additional ingredientsin a pharmaceutical composition provided herein will vary, dependingupon the identity, size, and/or condition of the subject treated andfurther depending upon the route by which the composition is to beadministered. By way of example, the composition can comprise between0.1% and 100% (w/w) of the compound of formula (I).

In some embodiments, the pharmaceutically acceptable excipient is atleast 95%, 96%, 97%, 98%, 99%, or 100% pure. In some embodiments, theexcipient is approved for use in humans and for veterinary use. In someembodiments, the excipient has been approved by United States Food andDrug Administration. In some embodiments, the excipient ispharmaceutical grade. In some embodiments, the excipient meets thestandards of the United States Pharmacopoeia (USP), the EuropeanPharmacopoeia (EP), the British Pharmacopoeia, and/or the InternationalPharmacopoeia.

Pharmaceutically acceptable excipients used in the manufacture ofpharmaceutically acceptable compositions include, but are not limitedto, inert diluents, dispersing and/or granulating agents, surface activeagents and/or emulsifiers, disintegrating agents, binding agents,preservatives, buffering agents, lubricating agents, and/or oils. One ormore such excipients can optionally be included in the formulations.Excipients such as cocoa butter and suppository waxes, coloring agents,coating agents, sweetening, flavoring, and perfuming agents can bepresent in the pharmaceutically acceptable composition, according to thejudgment of the formulator.

Exemplary pharmaceutically acceptable excipients include, but are notlimited to, diluents such as calcium carbonate, sodium carbonate,calcium phosphate, dicalcium phosphate, calcium sulfate, calciumhydrogen phosphate, sodium phosphate lactose, sucrose, cellulose,microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodiumchloride, dry starch, cornstarch, powdered sugar, etc., and combinationsthereof

Exemplary granulating and/or dispersing agents include, but are notlimited to, potato starch, corn starch, tapioca starch, sodium starchglycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite,cellulose and wood products, natural sponge, cation-exchange resins,calcium carbonate, silicates, sodium carbonate, cross-linkedpoly(vinyl-pyrrolidone) (crospovidone), sodium carboxymethyl starch(sodium starch glycolate), carboxymethyl cellulose, cross-linked sodiumcarboxymethyl cellulose (croscarmellose), methylcellulose,pregelatinized starch (starch 1500), microcrystalline starch, waterinsoluble starch, calcium carboxymethyl cellulose, magnesium aluminumsilicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds,etc., and combinations thereof.

Exemplary surface active agents and/or emulsifiers include, but are notlimited to, natural emulsifiers (e.g. acacia, agar, alginic acid, sodiumalginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin,egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidalclays (e.g. bentonite [aluminum silicate] and Veegum [magnesium aluminumsilicate]), long chain amino acid derivatives, high molecular weightalcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetinmonostearate, ethylene glycol distearate, glyceryl monostearate, andpropylene glycol monostearate, polyvinyl alcohol), carbomers (e.g.carboxy polymethylene, polyacrylic acid, acrylic acid polymer, andcarboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g.carboxymethylcellulose sodium, powdered cellulose, hydroxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose,methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylenesorbitan monolaurate [Tween 20], polyoxyethylene sorbitan [Tween 60],polyoxyethylene sorbitan monooleate [Tween 80], sorbitan monopalmitate[Span 40], sorbitan monostearate [Span 60], sorbitan tristearate [Span65], glyceryl monooleate, sorbitan monooleate [Span 80]),polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj 45],polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil,polyoxymethylene stearate, and Solutol), sucrose fatty acid esters,polyethylene glycol fatty acid esters (e.g. Cremophor), polyoxyethyleneethers, (e.g. polyoxyethylene lauryl ether [Brij 30]),poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamineoleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyllaurate, sodium lauryl sulfate, Pluronic F 68, Poloxamer 188,cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride,docusate sodium, etc. and/or combinations thereof.

Exemplary binding agents include, but are not limited to, starch (e.g.cornstarch and starch paste); gelatin; sugars (e.g. sucrose, glucose,dextrose, dextrin, molasses, lactose, lactitol, mannitol, etc.); naturaland synthetic gums (e.g. acacia, sodium alginate, extract of Irish moss,panwar gum, ghatti gum, mucilage of isapol husks,carboxymethylcellulose, methylcellulose, ethylcellulose,hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate,poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum), and larcharabogalactan); alginates; polyethylene oxide; polyethylene glycol;inorganic calcium salts; silicic acid; polymethacrylates; waxes; water;alcohol; etc.; and combinations thereof.

Exemplary preservatives can include antioxidants, chelating agents,antimicrobial preservatives, antifungal preservatives, alcoholpreservatives, acidic preservatives, and other preservatives. Exemplaryantioxidants include, but are not limited to, alpha tocopherol, ascorbicacid, acorbyl palmitate, butylated hydroxyanisole, butylatedhydroxytoluene, monothioglycerol, potassium metabisulfite, propionicacid, propyl gallate, sodium ascorbate, sodium bisulfite, sodiummetabisulfite, and sodium sulfite. Exemplary chelating agents includeethylenediaminetetraacetic acid (EDTA), citric acid monohydrate,disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malicacid, phosphoric acid, sodium edetate, tartaric acid, and trisodiumedetate. Exemplary antimicrobial preservatives include, but are notlimited to, benzalkonium chloride, benzethonium chloride, benzylalcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine,chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol,glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethylalcohol, phenylmercuric nitrate, propylene glycol, and thimerosal.Exemplary antifungal preservatives include, but are not limited to,butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoicacid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodiumbenzoate, sodium propionate, and sorbic acid. Exemplary alcoholpreservatives include, but are not limited to, ethanol, polyethyleneglycol, phenol, phenolic compounds, bisphenol, chlorobutanol,hydroxybenzoate, and phenylethyl alcohol. Exemplary acidic preservativesinclude, but are not limited to, vitamin A, vitamin C, vitamin E,beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbicacid, sorbic acid, and phytic acid. Other preservatives include, but arenot limited to, tocopherol, tocopherol acetate, deteroxime mesylate,cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened(BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ethersulfate (SLES), sodium bisulfite, sodium metabisulfite, potassiumsulfite, potassium metabisulfite, Glydant Plus, Phenonip, methylparaben,Germall 115, Germaben II, Neolone, Kathon, and Euxyl. In certainembodiments, the preservative is an anti-oxidant. In other embodiments,the preservative is a chelating agent.

Exemplary buffering agents include, but are not limited to, citratebuffer solutions, acetate buffer solutions, phosphate buffer solutions,ammonium chloride, calcium carbonate, calcium chloride, calcium citrate,calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconicacid, calcium glycerophosphate, calcium lactate, propanoic acid, calciumlevulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid,tribasic calcium phosphate, calcium hydroxide phosphate, potassiumacetate, potassium chloride, potassium gluconate, potassium mixtures,dibasic potassium phosphate, monobasic potassium phosphate, potassiumphosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride,sodium citrate, sodium lactate, dibasic sodium phosphate, monobasicsodium phosphate, sodium phosphate mixtures, tromethamine, magnesiumhydroxide, aluminum hydroxide, alginic acid, pyrogen-free water,isotonic saline, Ringer's solution, ethyl alcohol, etc., andcombinations thereof.

Exemplary lubricating agents include, but are not limited to, magnesiumstearate, calcium stearate, stearic acid, silica, talc, malt, glycerylbehanate, hydrogenated vegetable oils, polyethylene glycol, sodiumbenzoate, sodium acetate, sodium chloride, leucine, magnesium laurylsulfate, sodium lauryl sulfate, etc., and combinations thereof.

Exemplary oils include, but are not limited to, almond, apricot kernel,avocado, babassu, bergamot, black current seed, borage, cade, camomile,canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, codliver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose,fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop,isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon,litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink,nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel,peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary,safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, sheabutter, silicone, soybean, sunflower, tea tree, thistle, tsubaki,vetiver, walnut, and wheat germ oils. Exemplary oils include, but arenot limited to, butyl stearate, caprylic triglyceride, caprictriglyceride, cyclomethicone, diethyl sebacate, dimethicone 360,isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol,silicone oil, and combinations thereof.

Liquid dosage forms for oral and parenteral administration include, butare not limited to, pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto the compound of formula (I), the liquid dosage forms can compriseinert diluents commonly used in the art such as, for example, water orother solvents, solubilizing agents and emulsifiers such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils (in particular, cottonseed, groundnut, corn,germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions caninclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, and perfuming agents. In certainembodiments for parenteral administration, the conjugates providedherein are mixed with solubilizing agents such as Cremophor, alcohols,oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, andcombinations thereof. For example, in certain embodiments, the oralsuspension can comprise at least one compound of formula (I) andcarboxymethylcellulose. In some embodiments, the oral suspension cancomprise at least one compound of formula (I), carboxymethylcellulose,and DMSO. In one embodiment, the oral suspension can comprise a compoundof formula (I) and 0.5% carboxymethylcellulose/5% DMSO/0.5% Tween(PKPD#5). In another embodiment, the oral suspension can comprise acompound of formula (I) and between about 0.1 and 2%carboxymethylcellulose.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions can be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation can be a sterile injectable solution,suspension or emulsion in a nontoxic parenterally acceptable diluent orsolvent, for example, as a solution in 1,3-butanediol. Among theacceptable vehicles and solvents that can be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use. Injectable compositions can contain from about 0.1to about 5% w/w of the compound of formula (I).

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This can be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolutionwhich, in turn, can depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform can be accomplished by dissolving or suspending the drug in an oilvehicle.

Compositions for rectal or vaginal administration are typicallysuppositories which can be prepared by mixing the conjugates providedherein with suitable non-irritating excipients such as cocoa butter,polyethylene glycol or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the compound of formula (I).

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the compoundof formula (I) is mixed with at least one inert, pharmaceuticallyacceptable excipient such as sodium citrate or dicalcium phosphateand/or a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form can comprise buffering agents. The unit dose formulation,for example, a tablet, can contain from about 0.05% to about 95% byweight of the compound of formula (I).

Solid compositions of a similar type can be employed as fillers in softand hard-filled gelatin capsules using such pharmaceutically acceptableexcipients as lactose or milk sugar as well as high molecular weightpolyethylene glycols and the like. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings and other coatings well known in thepharmaceutical formulating art. They can optionally comprise opacifyingagents and can be of a composition that they release the compound offormula (I) only. In some embodiments, the compound of formula (I) canbe released in a certain part of the intestinal tract, optionally, in adelayed manner. Examples of embedding compositions which can be usedinclude polymeric substances and waxes. Solid compositions of a similartype can be employed as fillers in soft and hard-filled gelatin capsulesusing such excipients as lactose or milk sugar as well as high molecularweight polethylene glycols and the like.

The compound of formula (I) can be in micro-encapsulated form with oneor more pharmaceutically acceptable excipients as noted above. In suchsolid dosage forms, the compound of formula (I) can be admixed with atleast one inert diluent such as sucrose, lactose or starch. Such dosageforms can comprise, as is normal practice, additional substances otherthan inert diluents, e.g., tableting lubricants and other tableting aidssuch a magnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms can comprise bufferingagents.

Dosage forms for topical and/or transdermal administration of a compoundof formula (I) provided herein can include ointments, pastes, creams,lotions, gels, powders, solutions, sprays, inhalants and/or patches.Generally, the compound of formula (I) is admixed under sterileconditions with one or more pharmaceutically acceptable excipientsand/or any needed preservatives and/or buffers as may be required.Additionally, the use of transdermal patches, which often have the addedadvantage of providing controlled delivery of a compound of formula (I)to the body, is contemplated herein. Such dosage forms can be prepared,for example, by dissolving and/or dispensing the compound of formula (I)in the proper medium. Alternatively or additionally, the rate can becontrolled by either providing a rate controlling membrane and/or bydispersing the compound of formula (I) in a polymer matrix and/or gel.

Suitable devices for use in delivering intradermal pharmaceuticallyacceptable compositions described herein include short needle devicessuch as those described in U.S. Pat. Nos. 4,886,499; 5,190,521;5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662.Intradermal compositions can be administered by devices which limit theeffective penetration length of a needle into the skin, such as thosedescribed in PCT publication WO 99/34850 and functional equivalentsthereof. Jet injection devices which deliver liquid vaccines to thedermis via a liquid jet injector and/or via a needle which pierces thestratum corneum and produces a jet which reaches the dermis aresuitable. Jet injection devices are described, for example, in U.S. Pat.Nos. 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189;5,704,911; 5,383,851; 5,893,397; 5,466,220; 5,339,163; 5,312,335;5,503,627; 5,064,413; 5,520,639; 4,596,556; 4,790,824; 4,941,880;4,940,460; and PCT publications WO 97/37705 and WO 97/13537. Ballisticpowder/particle delivery devices which use compressed gas to acceleratevaccine in powder form through the outer layers of the skin to thedermis are suitable. Alternatively or additionally, conventionalsyringes can be used in the classical mantoux method of intradermaladministration.

Formulations suitable for topical administration include, but are notlimited to, liquid and/or semi liquid preparations such as liniments,lotions, oil in water and/or water in oil emulsions such as creams,ointments and/or pastes, and/or solutions and/or suspensions.Topically-administrable formulations can, for example, comprise fromabout 1% to about 10% (w/w) compound of formula (I), although theconcentration of the compound of formula (I) can be as high as thesolubility limit of the compound of formula (I) in the solvent. In someembodiments, topically-administrable formulations can, for example,comprise from about 1% to about 9% (w/w) compound of formula (I), suchas from about 1% to about 8% (w/w), further such as from about 1% toabout 7% (w/w), further such as from about 1% to about 6% (w/w), furthersuch as from about 1% to about 5% (w/w), further such as from about 1%to about 4% (w/w), further such as from about 1% to about 3% (w/w), andfurther such as from about 1% to about 2% (w/w) compound of formula (I).Formulations for topical administration can further comprise one or moreof the additional pharmaceutically acceptable excipients describedherein.

A pharmaceutical composition provided herein can be prepared, packaged,and/or sold in a formulation suitable for pulmonary administration viathe buccal cavity. Such a formulation can comprise dry particles whichcomprise the compound of formula (I) and which have a diameter in therange from about 0.5 to about 7 nanometers, such as from about 1 toabout 6 nanometers, further such as from about 2 to about 5 nanometers,and further such as from about 3 to about 4 nanometers. Suchpharmaceutical compositions are conveniently in the form of dry powdersfor administration using a device comprising a dry powder reservoir towhich a stream of propellant can be directed to disperse the powderand/or using a self propelling solvent/powder dispensing container suchas a device comprising the compound of formula (I) dissolved and/orsuspended in a low-boiling propellant in a sealed container. Suchpowders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers.Alternatively, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositions caninclude a solid fine powder diluent such as sugar and can be provided ina unit dose form.

Low boiling propellants generally include liquid propellants having aboiling point of below 65° F. at atmospheric pressure. Generally, thepropellant can constitute 50% to 99.9% (w/w) of the pharmaceuticalcomposition, and the active ingredient can constitute 0.1% to 20% (w/w)of the pharmaceutical composition. The propellant can further compriseadditional excipients such as a liquid non-ionic and/or solid anionicsurfactant and/or a solid diluent (which may have a particle size of thesame order as particles comprising the compound of formula (I)).

Pharmaceutical compositions provided herein formulated for pulmonarydelivery can provide the compound of formula (I) in the form of dropletsof a solution and/or suspension. Such formulations can be prepared,packaged, and/or sold as aqueous and/or dilute alcoholic solutionsand/or suspensions, optionally sterile, comprising the compound offormula (I), and can be administered using any nebulization and/oratomization device. Such formulations can further comprise one or moreadditional excipients including, but not limited to, a flavoring agentsuch as saccharin sodium, a volatile oil, a buffering agent, a surfaceactive agent, and/or a preservative such as methylhydroxybenzoate. Thedroplets provided by this route of administration can have an averagediameter in the range from about 0.1 to about 200 nanometers.

The formulations described herein as being useful for pulmonary deliveryare useful for intranasal delivery of a pharmaceutical compositionprovided herein. Another formulation suitable for intranasaladministration is a coarse powder comprising the compound of formula (I)and having an average particle from about 0.2 to 500 micrometers. Such aformulation is administered, for example, by rapid inhalation throughthe nasal passage from a container of the powder held close to thenostrils.

Formulations suitable for nasal administration can, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe compound of formula (I), and can comprise one or more of theadditional excipients described herein. A pharmaceutical compositionprovided herein can be prepared, packaged, and/or sold in a formulationsuitable for buccal administration. Such formulations can, for example,be in the form of tablets and/or lozenges made using conventionalmethods, and can, for example, comprise 0.1 to 20% (w/w) of the compoundof formula (I), the balance comprising an orally dissolvable and/ordegradable composition and, optionally, one or more of the additionalpharmaceutically acceptable excipients described herein. In someembodiments, formulations suitable for buccal administration cancomprise a powder and/or an aerosolized and/or atomized solution and/orsuspension comprising the compound of formula (I). Such powdered,aerosolized, and/or aerosolized formulations, when dispersed, can havean average particle and/or droplet size in the range from about 0.1 toabout 200 nanometers, and can further comprise one or more of theadditional pharmaceutically acceptable excipients described herein.

A pharmaceutical composition provided herein can be prepared, packaged,and/or sold in a formulation suitable for ophthalmic administration.Such formulations can, for example, be in the form of eye dropsincluding, for example, a 0.1/1.0% (w/w) solution and/or suspension ofthe compound of formula (I) in an aqueous or oily liquid carrier. Suchdrops can further comprise buffering agents, salts, and/or one or moreother of the additional pharmaceutically acceptable excipients describedherein. Other opthalmically-administrable formulations which are usefulinclude those which comprise the compound of formula (I) inmicrocrystalline form and/or in a liposomal preparation. Ear dropsand/or eye drops are contemplated as being within the scope of thisdisclosure.

General considerations in the formulation and/or manufacture ofpharmaceutical compositions can be found, for example, in Remington: TheScience and Practice of Pharmacy 21^(st) Ed., (Lippincott Williams &Wilkins, 2005).

Although the descriptions of pharmaceutical compositions provided hereinare principally directed to pharmaceutical compositions which aresuitable for administration to humans, it will be understood by theskilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and/or perform such modification with merely ordinary, if any,experimentation.

Further provided herein are kits comprising one or more compounds offormula (I) (or pharmaceutically acceptable forms thereof), and/or anpharmaceutical composition as described above. Kits are typicallyprovided in a suitable container (e.g., for example, a foil, plastic, orcardboard package). In certain embodiments, a kit can include one ormore pharmaceutically acceptable excipients, pharmaceutical additives,therapeutically active agents, and the like, as described herein. Incertain embodiments, a kit can include means for proper administration,such as, for example, graduated cups, syringes, needles, cleaning aids,and the like. In certain embodiments, a kit can include instructions forproper administration and/or preparation for proper administration.

The instructions would direct the consumer or medical personnel toadminister the dosage form according to administration modes known tothose skilled in the art. Such kits could be packaged and sold in singleor multiple kit units. An example of such a kit is a so-called blisterpack. Blister packs are well known in the packaging industry and arebeing widely used for the packaging of pharmaceutical unit dosage forms(tablets, capsules, and the like). Blister packs generally consist of asheet of relatively stiff material covered with a foil of a preferablytransparent plastic material. During the packaging process, recesses areformed in the plastic foil. The recesses have the size and shape of thetablets or capsules to be packed. Next, the tablets or capsules areplaced in the recesses and the sheet of relatively stiff material issealed against the plastic foil at the face of the foil which isopposite from the direction in which the recesses were formed. As aresult, the tablets or capsules are sealed in the recesses between theplastic foil and the sheet. The strength of the sheet is such that thetablets or capsules can be removed from the blister pack by manuallyapplying pressure on the recesses whereby an opening is formed in thesheet at the place of the recess. The tablet or capsule can then beremoved via said opening.

It can be desirable to provide a memory aid on the kit, e.g., in theform of numbers next to the tablets or capsules whereby the numberscorrespond with the days of the regimen which the tablets or capsules sospecified should be ingested. Another example of such a memory aid is acalendar printed on the card, e.g., as follows “First Week, Monday,Tuesday, . . . etc. . . . Second Week, Monday, Tuesday, . . . ” etc.Other variations of memory aids will be readily apparent. A “daily dose”can be a single tablet or capsule or several pills or capsules to betaken on a given day.

4. Uses and Methods of Treatment 4.1 Definitions

As used herein, and unless otherwise specified, the terms “treat,”“treating” and “treatment” contemplate an action that occurs while asubject is suffering from the specified disease, disorder or condition,which reduces the severity of the disease, disorder or condition, orretards or slows the progression of the disease, disorder or condition.

As used herein, unless otherwise specified, the terms “prevent,”“preventing” and “prevention” contemplate an action that occurs before asubject begins to suffer from the specified disease, disorder orcondition, which inhibits or reduces the severity of the disease,disorder or condition.

As used herein, and unless otherwise specified, the terms “manage,”“managing” and “management” encompass preventing the recurrence of thespecified disease, disorder or condition in a subject who has alreadysuffered from the disease, disorder or condition, and/or lengthening thetime that a subject who has suffered from the disease, disorder orcondition remains in remission. The terms encompass modulating thethreshold, development and/or duration of the disease, disorder orcondition, or changing the way that a subject responds to the disease,disorder or condition.

As used herein “inhibition”, “inhibiting”, “inhibit” and “inhibitor”,and the like, refer to the ability of a compound to reduce, slow, haltor prevent activity of a particular biological process (e.g., FASNactivity) in a cell relative to vehicle. In certain embodiments, theinhibition results in reduction of the activity by 5%, 10%, 15%, 20%,25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or more of the activitywithout such inhibition.

As used herein, and unless otherwise specified, a “therapeuticallyeffective amount” of a compound is an amount sufficient to provide atherapeutic benefit in the treatment or management of a disease,disorder or condition, or to delay or minimize one or more symptomsassociated with the disease, disorder or condition. A therapeuticallyeffective amount of a compound means an amount of therapeutic agent,alone or in combination with other therapies, which provides atherapeutic benefit in the treatment or management of the disease,disorder or condition. The term “therapeutically effective amount” canencompass an amount that improves overall therapy, reduces or avoidssymptoms or causes of disease or condition, or enhances the therapeuticefficacy of another therapeutic agent.

As used herein, and unless otherwise specified, a “prophylacticallyeffective amount” of a compound is an amount sufficient to prevent adisease, disorder or condition, or one or more symptoms associated withthe disease, disorder or condition, or prevent its recurrence. Aprophylactically effective amount of a compound means an amount oftherapeutic agent, alone or in combination with other agents, whichprovides a prophylactic benefit in the prevention of the disease,disorder or condition. The term “prophylactically effective amount” canencompass an amount that improves overall prophylaxis or enhances theprophylactic efficacy of another prophylactic agent.

4.2 Embodiments

In one embodiment, provided herein are methods for treating, preventingand/or managing a FASN-mediated disorder, disease or conditioncomprising administering to a subject in need thereof a therapeuticallyor prophylactically effective amount of at least one compound of formula(I) or a pharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof.

In another embodiment, provided herein are methods for inhibiting FASNin a subject comprising administering to a subject in need thereof atherapeutically effective amount of at least one compound of formula (I)or a pharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof.

In another embodiment, provided herein is a method of inhibitingactivation of the FASN pathway in vitro or ex vivo, comprisingcontacting a FASN protein with at least one compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof, in an amount sufficient toreduce the activation of the FASN pathway.

In another embodiment, provided herein is the use of at least onecompound of formula (I) or a pharmaceutically acceptable form thereof,or a pharmaceutical composition comprising at least one compound offormula (I) or a pharmaceutically acceptable form thereof, for thetreatment of a FASN-mediated disorder, disease or condition in asubject.

In another embodiment, provided herein is the use of at least onecompound of formula (I) or a pharmaceutically acceptable form thereof,or a pharmaceutical composition comprising at least one compound offormula (I) or a pharmaceutically acceptable form thereof, in themanufacture of a medicament. In certain embodiments, the medicament isuseful for treating a FASN-mediated disorder in a subject.

Compounds of formula (I) provided herein can be inhibitors of FASN. A“FASN-mediated disorder” as used herein, refers to a disease, disorderor condition which is treatable by inhibition of FASN activity.FASN-mediated disorders include, but are not limited to,hyperproliferative disorders; inflammatory disorders; obesity relateddisorders, such as, but not limited to, Type II diabetes mellitus andfatty liver disease; microbial infections, such as, but not limited to,viral, bacterial, fungal, parasitic, and protozoal infections; andcomplications thereof.

In certain embodiments, the FASN-mediated disorder is ahyperproliferative disorder. In certain embodiments, thehyperproliferative disorder is cancer. To date, aberrant FASN activityhas been observed in a variety of hyperproliferative disorders whichinclude, but are not limited to:

(i) bladder cancer (see Visca et al., Anticancer Res. (2003)23:335-339);

(ii) brain cancer (e.g., meningioma, see: Haase et al., Neuro-Oncology(2010) Advance Access published Feb. 5, 2010, 1-11; e.g., glioma: seeZhao et al., Br. J. Cancer (2006) 95:869-878; e.g., meduloblastoma: seeSlade et al., Anticancer Res. (2003) 23:1235-1243);

(iii) breast cancer (see Alo et al., Cancer (1996) 77:474-482; Pizer etal., Cancer Res. (1996) 56:2745-2747; Pizer et al., Cancer Res. (2000)60:213-218; Milgraum et al., Clin. Cancer Res. (1997) 3:2115-2120; Lupuand Menendez, Endocrinology (2006) 147:4056-4066; Alo et al., Oncol.Rep. (2000) 7:1383-1388; Wang et al., Cancer Lett. (2001) 167:99-104;Liu et al., Mol. Cancer Ther. (2008) 7:263-270; and Kuhajda et al., PNAS(2000) 97:3450-3454; e.g., mammary cancer: see Hennigar et al., Biochim.Biophys. Acta (1998) 1392:85-100 and Alli et al., Oncogene (2005)24:39-46);

(iv) colorectal cancer (see Rashid et al., Am. J. Pathol. (1997)150:201-208; Huang et al., World J. Gastroenterol. (2000) 6:295-297;Zhan et al., Clin. Cancer Res. (2008) 14:5735-5742);

(v) esophageal cancer (see Nemoto et al., Pathobiology (2001)69:297-303);

(vi) endometrial cancer (see Pizer et al., Cancer (1998) 83:528-537;Pizer et al., Int. J. Gynecol. Pathol. (1997) 16:45-51; Lupu andMenendez, Endocrinology (2006) 147:4056-406; and Sebastiani et al.,Gynecologic Oncology (2004) 92:101-105);

(viii) gastric cancer (see Kusakabe et al., Histopathology (2002)40:71-79);

(ix) gastrointestinal stromal tumor (see Rossi et al., J. Pathol. (2006)209:369-375);

(x) kidney cancer (e.g., nephroblastoma/Wilms' tumor: see Camassei etal., Med. Pediatr. Oncol. (2003) 40:302-308);

(xi) liver cancer (see Evert et al., Lab. Invest. (2005) 85:99-108);

(xii) lung cancer (see Piyathilake et al., Human Pathol. (2000)31:1068-1073 and Visca et al., Anticancer Res. (2004) 24:4169-4173);

(xiii) mesothelioma (see Gabrielson et al., Clin. Cancer Research (2001)7:153-157);

(xiv) multiple myeloma (see Wang et al., J. Zhejiang Univ. Sci B (2008)9:441-447);

(xv) neuroblastoma (see Slade et al., Anticancer Res. (2003)23:1235-1243);

(xvi) oral cancer (see Krontiras et al., Head Neck (1999) 21:325-329;and Agostini et al., Oral Oncol. (2004) 40:728-735; see also e.g., oralsquamous cell carcinoma (OSCC): Silva et al., Oral Diseases (2007)14:376-382);

(xvii) ovarian cancer (see Pizer et al., Cancer Res. (1996)56:1189-1193; Alo et al., Oncol. Rep. (2000) 7:1383-1388; Wang et al.,Oncogene (2005) 24:3574-3582; Gansler et al., Hum. Pathol. (1997)28:686-692; and Zhou et al., Cancer Res. (2007) 2964-2971);

(xviii) pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductalpapillary mucinous neoplasm (IPMN): see Walter et al., Cancer Epidemiol.Biomarkers Prev. (2009) 18:2380-2385);

(xix) Pagets disease of the vulva (see Alo et al., Int. J Gynecol.Pathol. (2005) 24:404-408);

(xx) prostate cancer (see Pizer et al., Proc Am. Assoc. Cancer Res.(2000) 41:655; Swinnen et al., Int. J Cancer (2002) 98:19-22; Epstein etal., Urology (1995) 45:81-86; De Schrijver et al., Cancer Res. (2003)63:3799-3804; Pizer et al., Prostate (2001) 47:102-110; Furuya et al.,Anticancer Res. (1997) 17:4589-4593; Shurbaji et al., Hum. Pathol.(1996) 27:917-921; Migita et al., J. Nat. Cancer Inst. (2009)101:519-532; Rossi et al., Mol. Cancer Res. (2003) 1:707-715; and Shahet al., Hum. Pathol. (2006) 37:401-409);

(xxi) retinoblastoma (see Camassei et al., Investig. Opthalmol. Vis.Sci. (2003) 44:2399-2403; and Slade et al., Anticancer Res. (2003)23:1235-1243);

(xxii) soft tissue sarcoma (e.g., malignant fibrous histiocytoma (MFH),liposarcoma, malignant peripheral nerve sheath tumor (MPNST),chondrosarcoma: see Takahiro et al., Clin. Cancer Res. (2003)9:2204-2212);

(xxiii) skin cancer (e.g., melanoma: see Innocenzi et al., J. Cutan.Pathol. (2003) 30:23-28; Kapur et al., Modern Pathology (2005)18:1107-1112 and Carvalho et al., Int. J. Cancer (2008) 123:2557-2565);and

(xxiv) thyroid cancer (see Vald et al., Mod. Path. (1999) 12:70A;Sekiguchi et al., Biomed. Pharmacother. (2001) 55:466-474; e.g.,papillary thyroid carcinoma (PTC): see Uddin et al., J. Clin.Endocrinol. Metab. (2008) 93:4088-4097).

It is envisioned that aberrant FASN activity plays a role in otherhyperproliferative disorders. Exemplary hyperproliferative diseases,disorders, conditions or cancers include, but are not limited to,acoustic neuroma, adenocarcinoma, adrenal gland cancer, angiosarcoma(e.g., lymphangiosarcoma, lymphangioendotheliosarcoma, hemangiosarcoma),benign monoclonal gammopathy, biliary cancer (e.g., cholangiocarcinoma),bladder cancer, breast cancer (e.g., adenocarcinoma of the breast,papillary carcinoma of the breast, mammary cancer, medullary carcinomaof the breast), brain cancer (e.g., meningioma; glioma, e.g.,astrocytoma, oligodendroglioma; medulloblastoma), bronchus cancer (e.g.,bronchogenic carcinoma), cervical cancer (e.g., cervicaladenocarcinoma), choriocarcinoma, chordoma, craniopharyngioma,colorectal cancer (e.g., colorectal adenocarcinoma), epithelialcarcinoma, ependymoma, endotheliosarcoma (e.g., Kaposi's sarcoma,multiple idiopathic hemorrhagic sarcoma), endometrial cancer, esophagealcancer (e.g., adenocarcinoma of the esophagus, Barrett's adenocarinoma),Ewing sarcoma, familiar hypereosinophilia, gastric cancer (e.g., stomachadenocarcinoma), gastrointestinal stromal tumor (GIST), head and neckcancer, heavy chain disease (e.g., alpha chain disease, gamma chaindisease, mu chain disease), hemangioblastoma, inflammatorymyofibroblastic tumors, immunocytic amyloidosis, kidney cancer (e.g.,nephroblastoma a.k.a. Wilms' tumor, renal cell carcinoma), liver cancer(e.g., hepatocellular cancer (HCC) such as hepatocellular carcinoma,malignant hepatoma), lung cancer (e.g., small cell lung cancer (SCLC),non-small cell lung cancer (NSCLC), adenocarcinoma of the lung),leukemia (e.g., acute lymphocytic leukemia (ALL), acute myelocyticleukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocyticleukemia (CLL)), lymphoma (e.g., Hodgkin lymphoma, non-Hodgkin lymphoma(NHL), follicular lymphoma, diffuse large B-cell lymphoma (DLBCL),mantle cell lymphoma (MCL)), leiomyosarcoma (LMS), mastocytosis (e.g.,systemic mastocytosis), multiple myeloma (MM), myelodysplastic syndrome(MDS), mesothelioma, myeloproliferative disorder (MPD) (e.g.,polycythemia Vera (PV), essential thrombocytosis (ET), agnogenic myeloidmetaplasia (AMM) a.k.a. myelofibrosis (MF), chronic idiopathicmyelofibrosis, chronic myelocytic leukemia (CML), chronic neutrophilicleukemia (CNL), hypereosinophilic syndrome (HES)), neuroblastoma,neurofibroma (e.g., neurofibromatosis (NF) type 1 or type 2,schwannomatosis), neuroendocrine cancer (e.g., gastroenteropancreaticneuroendoctrine tumor (GEP-NET), carcinoid tumor), osteosarcoma, oralcancer (e.g., oral squamous cell carcinoma (OSCC)), ovarian cancer(e.g., cystadenocarcinoma, ovarian embryonal carcinoma, ovarianadenocarcinoma), Paget's disease of the vulva, Paget's disease of thepenis, papillary adenocarcinoma, pancreatic cancer (e.g., pancreaticandenocarcinoma, intraductal papillary mucinous neoplasm (IPMN)),pinealoma, primitive neuroectodermal tumor (PNT), prostate cancer (e.g.,prostate adenocarcinoma), rhabdomyosarcoma, retinoblastoma, salivarygland cancer, skin cancer (e.g., squamous cell carcinoma (SCC),keratoacanthoma (KA), melanoma, basal cell carcinoma), small bowelcancer (e.g., appendix cancer), soft tissue sarcoma (e.g., malignantfibrous histiocytoma (MFH), liposarcoma, malignant peripheral nervesheath tumor (MPNST), chondrosarcoma, fibrosarcoma, myxosarcoma),sebaceous gland carcinoma, sweat gland carcinoma, synovioma, testicularcancer (e.g., seminoma, testicular embryonal carcinoma), thyroid cancer(e.g., papillary carcinoma of the thyroid, papillary thyroid carcinoma(PTC), medullary thyroid cancer), and Waldenstrom's macroglobulinemia.

In certain embodiments, the hyperproliferative disorder is selected frombladder cancer, brain cancer, breast cancer, colorectal cancer,esophageal cancer, endometrial cancer, gastric cancer, gastrointestinalstromal tumor, kidney cancer, liver cancer, lung cancer, mesothelioma,multiple myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreaticcancer, prostate cancer, Paget's disease of the vulva, retinoblastoma,soft tissue sarcoma, skin cancer or thyroid cancer.

In certain embodiments, the cancer is selected from mesothelioma,multiple myeloma, neuroblastoma, Paget's disease, retinoblastoma,leukemia, myelodisplastic syndrome, or soft tissue sarcoma.

In certain embodiments, the brain cancer is meningioma, glioma ormeduloblastoma.

In certain embodiments, the oral cancer is oral squamous cell carcinoma.

In certain embodiments, the pancreatic cancer is pancreaticandenocarcinoma or intraductal papillary mucinous neoplasm.

In certain embodiments, the soft tissue carcinoma is malignant fibroushistiocytoma, liposarcoma, malignant peripheral nerve sheath tumor, orchondrosarcoma.

In certain embodiments, the skin cancer is melanoma.

In certain embodiments, the thyroid cancer is papillary thyroidcarcinoma.

In certain embodiments, the FASN-mediated disorder is an inflammatorydisorder. The term “inflammatory disorder” refers to a disease orcondition characterized by one or more symptoms of pain, heat, redness,swelling, and loss of function. Inflammatory disorders are meant toencompass inflammation associated with immune system disorders as wellas inflammation associated with non-immune system disorders.Inflammatory disorders are meant to encompass acute inflammation andchronic inflammation. To date, aberrant FASN activity has been observedin inflammatory bowel diseases such as ulcerative colitis (seeConsolazio et al., Anatomic Pathology (2006) 126:113-118; Rashid et al.,Am. J. Pathol. (1997) 150:201-208). It is envisioned that aberrant FASNactivity plays a role in other inflammatory disorders.

Exemplary inflammatory disorders include, but are not limited to,inflammation associated with acne, anemia (e.g., aplastic anemia,haemolytic autoimmune anaemia), asthma, arteritis (e.g., polyarteritis,temporal arteritis, periarteritis nodosa, Takayasu's arteritis),arthritis (e.g., crystalline arthritis, osteoarthritis, psoriaticarthritis, gouty arthritis, reactive arthritis, rheumatoid arthritis andReiter's arthritis), ankylosing spondylitis, amylosis, amyotrophiclateral sclerosis, autoimmune diseases, allergies or allergic reactions,Alzheimer's disease, atherosclerosis, bronchitis, bursitis, cancer,chronic prostatitis, conjunctivitis, Chagas disease, chronic obstructivepulmonary disease, cermatomyositis, diverticulitis, diabetes (e.g., typeI diabetes mellitus, type 2 diabetes mellitus), dermatitis, eosinophilicgastrointestinal disorders (e.g., eosinophilic esophagitis, eosinophilicgastritis, eosinophilic gastroenteritis, eosinophilic colitis), eczema,endometriosis, gastrointestinal bleeding, gastritis, gastroesophagealreflux disease (GORD, or its synonym GERD), Guillain-Barre syndrome,infection, ischaemic heart disease, Kawasaki disease,glomerulonephritis, gingivitis, hypersensitivity, headaches (e.g.,migraine headaches, tension headaches), ileus (e.g., postoperative ileusand ileus during sepsis), idiopathic thrombocytopenic purpura,interstitial cystitis, inflammatory bowel disease (IBD) (e.g., Crohn'sdisease, ulcerative colitis, collagenous colitis, lymphocytic colitis,ischaemic colitis, diversion colitis, Behcet's syndrome, indeterminatecolitis), inflammatory bowel syndrome (IBS), lupus, multiple sclerosis,morphea, myeasthenia gravis, myocardial ischemia, nephrotic syndrome,pemphigus vulgaris, pernicious aneaemia, peptic ulcers, psoriasis,polymyositis, primary biliary cirrhosis, Parkinson's disease, pelvicinflammatory disease, reperfusion injury, regional enteritis, rheumaticfever, systemic lupus erythematosus, schleroderma, scierodoma,sarcoidosis, spondyloarthopathies, Sjogren's syndrome, thyroiditis,transplantation rejection, tendonitis, trauma or injury (e.g.,frostbite, chemical irritants, toxins, scarring, burns, physicalinjury), vasculitis, vitiligo and Wegener's granulomatosis.

In some embodiment, the inflammatory disorder is selected from anemia,asthma, arteritis, arthritis, chronic obstructive pulmpnary disease,dermatitis, gastroesophageal reflux disease, Crohn's disease,inflammatory bowel syndrome, multiple sclerosis, psoriasis and anautoimmune disease.

Inhibition of FASN activity has also been observed to reduce body weight(e.g., by blocking the body's ability to convert carbohydrates to fat)and to suppress appetite (see Loftus et al., Science (2000)288:2379-2381). Reduction of storage fat is expected to provide variousprimary and/or secondary benefits in a subject (e.g., in a subjectdiagnosed with a complication associated with obesity) such as, forexample, an increased insulin responsiveness (e.g., in a subjectdiagnosed with Type II diabetes mellitus); a reduction in elevated bloodpressure; a reduction in elevated cholesterol levels; and/or a reduction(or a reduced risk or progression) of ischemic heart disease, arterialvascular disease, angina, myocardial infarction, stroke, migraines,congestive heart failure, deep vein thrombosis, pulmonary embolism, gallstones, gastroesophagael reflux disease, obstructive sleep apnea,obesity hypoventilation syndrome, asthma, gout, poor mobility, backpain, erectile dysfunction, urinary incontinence, liver injury (e.g.,fatty liver disease, liver cirrhosis, alcoholic cirrhosis, endotoxinmediated liver injury) or chronic renal failure. Thus, In someembodiments, disclosed methods are applicable to obese subjects,diabetic subjects, and alcoholic subjects, and are generally useful aspart of a program to treat an obesity-related disorder or a complicationthereof.

An “obesity-related disorder” as used herein, includes, but is notlimited to, obesity, undesired weight gain (e.g., frommedication-induced weight gain, from cessation of smoking) and anover-eating disorder (e.g., binge eating, bulimia, compulsive eating, ora lack of appetite control each of which can optionally lead toundesired weight gain or obesity). “Obesity” and “obese” as used herein,refers to class I obesity, class II obesity, class III obesity orpre-obesity (e.g., being “over-weight”) as defined by the World HealthOrganization.

In some embodiments, obesity-related disorder include, but are notlimited to, Type II diabetes mellitus, elevated blood pressure, elevatedcholesterol levels, ischemic heart disease, arterial vascular disease,angina, myocardial infarction, stroke, migraines, congestive heartfailure, deep vein thrombosis, pulmonary embolism, gall stones,gastroesophagael reflux disease, obstructive sleep apnea, obesityhypoventilation syndrome, asthma, gout, poor mobility, back pain,erectile dysfunction, urinary incontinence, liver injury, fatty liver,and chronic renal failure.

In some embodiments, treatment of an obesity-related disorder orcomplication thereof involves reduction of body weight in the subject.In some embodiments, treatment of an obesity-related disorder orcomplication thereof involves appetite control in the subject.

In other embodiments, provided herein are methods for treating,preventing and/or managing a microbial infection (e.g., such as abacterial infection, viral infection, fungal infection, or parasitic orprotozoal infection) comprising administering to a subject atherapeutically or prophylactically effective amount of at least onecompound of formula (I) or a pharmaceutically acceptable form thereof,or a pharmaceutical composition comprising at least one compound offormula (I) or a pharmaceutically acceptable form thereof.

Also provided herein is the use of at least one compound of formula (I),or a pharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof, for the treatment, preventionand/or management of a microbial infection in a subject.

Also provided herein is the use of at least one compound of formula (I),or a pharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof, in the manufacture of amedicament useful for treating, preventing and/or managing a microbialinfection.

FASN has been identified as a target for treatment of microbialinfections, e.g., such as a viral infection, for example, infection withan enveloped virus such as the herpes virus (e.g., human cytomegalomousvirus (HCMV), herpes simplex virus 1 (HSV-1), herpes simplex virus 2(HSV-2), varicella zoster virus (VZV), Epstein-Barr virus), influenza Avirus and Heptatitis C virus (HCV) (see Munger et al., NatureBiotechnology (2008) 26: 1179-1186; Syed et al., Trends in Endocrinologyand Metabolism (2009) 21:33-40; Sakamoto et al., Nature Chemcial Biology(2005) 1:333-337; Yang et al., Hepatology (2008) 48:1396-1403) or apicornavirus such as Coxsackievirus B3 (CVB3) (see Rassmann et al.,Anti-viral Research (2007) 76:150-158). Other exemplary viruses include,but are not limited to, the hepatitis B virus, HIV, poxvirus,hepadavirus, retrovirus, and RNA viruses such as flavivirus, togavirus,coronavirus, Hepatitis D virus, orthomyxovirus, paramyxovirus,rhabdovirus, bunyavirus, and filovirus.

In some embodiments, the virus infects humans. In other embodiments, thevirus infects non-human animals. In another embodiment, the virusinfects primates (e.g., cynomolgus monkeys, rhesus monkeys); mammals,including commercially relevant mammals such as cattle, pigs, horses,sheep, goats, cats, and/or dogs; and/or birds, including commerciallyrelevant birds such as chickens, ducks, geese, and/or turkeys.

In certain embodiments, the virus is an enveloped virus. Examplesinclude, but are not limited to, viruses that are members of thehepadnavirus family, herpesvirus family, iridovirus family, poxvirusfamily, flavivirus family, togavirus family, retrovirus family,coronavirus family, filovirus family, rhabdovirus family, bunyavirusfamily, orthomyxovirus family, paramyxovirus family, and arenavirusfamily. Other examples include, but are not limited to, Hepadnavirushepatitis B virus (HBV), woodchuck hepatitis virus, ground squirrel(Hepadnaviridae) hepatitis virus, duck hepatitis B virus, heronhepatitis B virus, Herpesvirus herpes simplex virus (HSV) types 1 and 2,varicella-zoster virus, cytomegalovirus (CMV), human cytomegalovirus(HCMV), mouse cytomegalovirus (MCMV), guinea pig cytomegalovirus(GPCMV), Epstein-Barr virus (EBV), human herpes virus 6 (HHV variants Aand B), human herpes virus 7 (HHV-7), human herpes virus 8 (HHV-8),Kaposi's sarcoma-associated herpes virus (KSHV), B virus Poxvirusvaccinia virus, variola virus, smallpox virus, monkeypox virus, cowpoxvirus, camelpox virus, ectromelia virus, mousepox virus, rabbitpoxviruses, raccoonpox viruses, molluscum contagiosum virus, orf virus,milker's nodes virus, bovin papullar stomatitis virus, sheeppox virus,goatpox virus, lumpy skin disease virus, fowlpox virus, canarypox virus,pigeonpox virus, sparrowpox virus, myxoma virus, hare fibroma virus,rabbit fibroma virus, squirrel fibroma viruses, swinepox virus, tanapoxvirus, Yabapox virus, Flavivirus dengue virus, hepatitis C virus (HCV),GB hepatitis viruses (GBV-A, GBV-B and GBV-C), West Nile virus, yellowfever virus, St. Louis encephalitis virus, Japanese encephalitis virus,Powassan virus, tick-borne encephalitis virus, Kyasanur Forest diseasevirus, Togavirus, Venezuelan equine encephalitis (VEE) virus,chikungunya virus, Ross River virus, Mayaro virus, Sindbis virus,rubella virus, Retrovirus human immunodeficiency virus (HIV) types 1 and2, human T cell leukemia virus (HTLV) types 1, 2, and 5, mouse mammarytumor virus (MMTV), Rous sarcoma virus (RSV), lentiviruses, Coronavirus,severe acute respiratory syndrome (SARS) virus, Filovirus Ebola virus,Marburg virus, Metapneumoviruses (MPV) such as human metapneumovirus(HMPV), Rhabdovirus rabies virus, vesicular stomatitis virus,Bunyavirus, Crimean-Congo hemorrhagic fever virus, Rift Valley fevervirus, La Crosse virus, Hantaan virus, Orthomyxovirus, influenza virus(types A, B, and C), Paramyxovirus, parainfluenza virus (PIV types 1, 2and 3), respiratory syncytial virus (types A and B), measles virus,mumps virus, Arenavirus, lymphocytic choriomeningitis virus, Juninvirus, Machupo virus, Guanarito virus, Lassa virus, Ampari virus, Flexalvirus, Ippy virus, Mobala virus, Mopeia virus, Latino virus, Paranavirus, Pichinde virus, Punta toro virus (PTV), Tacaribe virus andTamiami virus.

In some embodiments, the virus is a non-enveloped virus, i.e., the virusdoes not have an envelope and is naked. Examples include, but are notlimited to, viruses that are members of the parvovirus family,circovirus family, polyoma virus family, papillomavirus family,adenovirus family, iridovirus family, reovirus family, birnavirusfamily, calicivirus family, and picornavirus family. Specific examplesinclude, but are not limited to, canine parvovirus, parvovirus B19,porcine circovirus type 1 and 2, BFDV (Beak and Feather Disease virus,chicken anaemia virus, Polyomavirus, simian virus 40 (SV40), JC virus,BK virus, Budgerigar fledgling disease virus, human papillomavirus,bovine papillomavirus (BPV) type 1, cotton tail rabbit papillomavirus,human adenovirus (HAdV-A, HAdV-B, HAdV-C, HAdV-D, HAdV-E, and HAdV-F),fowl adenovirus A, bovine adenovirus D, frog adenovirus, Reovirus, humanorbivirus, human coltivirus, mammalian orthoreovirus, bluetongue virus,rotavirus A, rotaviruses (groups B to G), Colorado tick fever virus,aquareovirus A, cypovirus 1, Fiji disease virus, rice dwarf virus, riceragged stunt virus, idnoreovirus 1, mycoreovirus 1, Birnavirus, bursaldisease virus, pancreatic necrosis virus, Calicivirus, swine vesicularexanthema virus, rabbit hemorrhagic disease virus, Norwalk virus,Sapporo virus, Picornavirus, human polioviruses (1-3), humancoxsackieviruses A1-22, 24 (CA1-22 and CA24, CA23 (echovirus 9)), humancoxsackieviruses (B1-6 (CB1-6)), human echoviruses 1-7, 9, 11-27, 29-33,vilyuish virus, simian enteroviruses 1-18 (SEV1-18), porcineenteroviruses 1-11 (PEV1-11), bovine enteroviruses 1-2 (BEV1-2),hepatitis A virus, rhinoviruses, hepatoviruses, cardioviruses,aphthoviruses and echoviruses.

In certain embodiments, the virus is a herpes virus, e.g., HSV-I, HSV-2,and CMV. In another embodiment, the virus is HCMV. In anotherembodiment, the virus is a liver trophic virus. In another embodiment,the virus is an influenza virus. In some embodiments, the virus is HIV.In certain embodiments, the virus is a hepatitis B virus. In a specificembodiment, the virus is EBV. In some embodiments, the virus is Kaposi'ssarcoma-associated herpes virus (KSHV). In certain embodiments the virusis a variola virus. In one embodiment, the virus is a Dengue virus. Inother embodiments, the virus is a SARS virus. In one embodiment, thevirus is an Ebola virus. In some embodiments the virus is a Marburgvirus. In certain embodiments, the virus is a measles virus. Inparticular embodiments, the virus is a vaccinia virus. In someembodiments, the virus is varicella-zoster virus (VZV). In someembodiments, the virus is a picornavirus. In certain embodiments thevirus is a rhinovirus. In certain embodiments the virus is not arhinovirus. In some embodiments, the virus is an adenovirus. Inparticular embodiments, the virus is a coxsackievirus (e.g.,coxsackievirus B3). In some embodiments, the virus is a rhinovirus. Incertain embodiments, the virus is a human papillomavirus (HPV).

In certain embodiments, the virus is a DNA virus. In other embodiments,the virus is an RNA virus. In one embodiment, the virus is a DNA or aRNA virus with a single-stranded genome. In another embodiment, thevirus is a DNA or a RNA virus with a double-stranded genome.

In some embodiments, the virus has a linear genome. In otherembodiments, the virus has a circular genome. In some embodiments, thevirus has a segmented genome. In other embodiments, the virus has anon-segmented genome.

In some embodiments, the virus is a positive-stranded RNA virus.

In other embodiments, the virus is a negative-stranded RNA virus. In oneembodiment, the virus is a segmented, negative-stranded RNA virus. Inanother embodiment, the virus is a non-segmented negative-stranded RNAvirus.

In some embodiments, the virus is an icosahedral virus. In otherembodiments, the virus is a helical virus. In yet other embodiments, thevirus is a complex virus.

In some embodiments, the virus is a hepatitis C virus.

In certain embodiments, the virus is selected from: a herpes virus suchas HSV-1, HSV-2, VZV, EBV, CMV (HCMV, MCMV, GPCMV), HMCV, CVB3, HHV-6and HHV-8; an influenza virus such as influenza type A and influenzatype B; respiratory viruses such as RSV, PIV (types 1, 2 and 3), measlesvirus, rhinovirus, adenovirus, HMPV and SARS virus; orthopoxviruses suchas vaccinia virus, cowpox virus, ectromelia virus, monkeypox virus andrabbitpox virus; a hepatitis virus such as HBV and HCV; a papova virussuch as papillomavirus (e.g., cotton tail rabbit papillomavirus andhuman papillomavirus) and BK virus; or other viruses such as VEE virus,Rift Valley fever virus, Tacaribe virus, Yellow fever virus, West Nilevirus, dengue virus, PTV and Pichinde virus.

In one embodiment, the virus is HSV-1. In another embodiment, the virusis HSV-2. In another embodiment, the virus is VZV. In anotherembodiment, the virus is EBV. In another embodiment, the virus is HCMV.In another embodiment, the virus is MCMV. In another embodiment, thevirus is GPCMV. In another embodiment, the virus is HHV-6. In anotherembodiment, the virus is HHV-8.

In one embodiment, the virus is influenza type A virus. In anotherembodiment, the virus is influenza type B virus.

In one embodiment, the virus is RSV. In another embodiment, the virus isPIV-3. In another embodiment, the virus is measles virus. In anotherembodiment, the virus is rhinovirus. In another embodiment, the virus isadenovirus. In another embodiment, the virus is HMPV. In anotherembodiment, the virus is SARS virus.

In one embodiment, the virus is vaccinia virus. In another embodiment,the virus is cowpox virus. In another embodiment, the virus isectromelia virus. In another embodiment, the virus is monkeypox virus.In another embodiment, the virus is rabbitpox virus.

In one embodiment, the virus is HBV. In another embodiment, the virus isHCV.

In one embodiment, the virus is cotton tail rabbit papillomavirus. Inanother embodiment, the virus is human papillomavirus. In anotherembodiment, the virus is BK virus.

In one embodiment, the virus is VEE virus. In another embodiment, thevirus is Rift Valley fever virus. In another embodiment, the virus isTacaribe virus. In another embodiment, the virus is Yellow fever virus.In another embodiment, the virus is West Nile virus. In anotherembodiment, the virus is dengue virus. In another embodiment, the virusis PTV. In another embodiment, the virus is Pichinde virus.

In certain embodiments, at least one compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can treat aninfection caused by one type of virus. In other embodiments, at leastone compound of formula (I) or a pharmaceutically acceptable formthereof, or a pharmaceutical composition comprising at least onecompound of formula (I) or a pharmaceutically acceptable form thereofprovided herein can treat one or more infections caused by two or moretypes of viruses at the same time. In other embodiments, at least onecompound of formula (I) or a pharmaceutically acceptable form thereof,or a pharmaceutical composition comprising at least one compound offormula (I) or a pharmaceutically acceptable form thereof providedherein can treat one or more infections caused by three or more types ofviruses at the same time. In other embodiments, at least one compound offormula (I) or a pharmaceutically acceptable form thereof, or apharmaceutical composition comprising at least one compound of formula(I) or a pharmaceutically acceptable form thereof provided herein cantreat one or more infections caused by four or more types of viruses atthe same time. In other embodiments, at least one compound of formula(I) or a pharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can treat oneor more infections caused by five or more types of viruses at the sametime. In other embodiments, at least one compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can treat oneor more infections caused by six, seven, eight, nine, ten, fifteen,twenty or more types of viruses at the same time.

In certain embodiments, the microbial infections can encompass thedisease related to infection by prions, e.g., scrapie, madcow disease,and any modified forms thereof. In certain embodiments, the microbialinfections encompass those prion diseases that affect humans.

It is envisioned that a compound of formula (I) or a pharmaceuticallyacceptable form thereof, or a pharmaceutical composition comprising atleast one compound of formula (I) or a pharmaceutically acceptable formthereof provided herein will also be useful in the treatment of othermicrobial infections, such as bacterial infections, fungal infections,and parasitic infections.

In certain embodiments, the microbial infection is a bacterialinfection. Examples of bacterial infections include, but are not limitedto, infections by mycobacteria (e.g., Mycobacteria tuberculosis, M.bovis, M. avium, M. leprae, and M. africanum), rickettsia, mycoplasma,chlamydia, and legionella. Other examples of bacterial infectionsinclude, but are not limited to, infections caused by Gram positivebacillus (e.g., Listeria, Bacillus such as Bacillus anthracis,Erysipelothrix species), Gram negative bacillus (e.g., Bartonella,Brucella, Campylobacter, Enterobacter, Escherichia, Francisella,Hemophilus, Klebsiella, Morganella, Proteus, Providencia, Pseudomonas,Salmonella, Serratia, Shigella, Vibrio and Yersinia species), spirochetebacteria (e.g., Borrelia species including Borrelia burgdorferi thatcauses Lyme disease), anaerobic bacteria (e.g., Actinomyces andClostridium species), Gram positive and negative coccal bacteria,Enterococcus species, Streptococcus species, Pneumococcus species,Staphylococcus species, and Neisseria species.

Specific examples of infectious bacteria include, but are not limitedto: Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria tuberculosis, M. avium, M. intracellulare, M. kansaii, M.gordonae, Staphylococcus aureus, Neisseria gonorrhoeae, Neisseriameningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group AStreptococcus), Streptococcus agalactiae (Group B Streptococcus),Streptococcus viridans, Streptococcus faecalis, Streptococcus bovis,Streptococcus pneumoniae, Haemophilus influenzae, Bacillus antracis,corynebacterium diphtheriae, Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasteurella multocida, Fusobacterium nucleatum,Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue,Leptospira, Rickettsia, and Actinomyces israelli.

In one embodiment, the bacterial infection is an infection caused byMycobacteria tuberculosis.

In certain embodiments, at least one compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can treat aninfection caused by one type of bacteria. In other embodiments, at leastone compound of formula (I) or a pharmaceutically acceptable formthereof, or a pharmaceutical composition comprising at least onecompound of formula (I) or a pharmaceutically acceptable form thereofprovided herein can treat one or more infections caused by two or moretypes of bateria at the same time. In other embodiments, at least onecompound of formula (I) or a pharmaceutically acceptable form thereof,or a pharmaceutical composition comprising at least one compound offormula (I) or a pharmaceutically acceptable form thereof providedherein can treat one or more infections caused by three or more types ofbacteria at the same time. In other embodiments, at least one compoundof formula (I) or a pharmaceutically acceptable form thereof, or apharmaceutical composition comprising at least one compound of formula(I) or a pharmaceutically acceptable form thereof provided herein cantreat one or more infections caused by four or more types of bacteria atthe same time. In other embodiments, at least one compound of formula(I) or a pharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can treat oneor more infections caused by five or more types of bacteria at the sametime. In other embodiments, at least one compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can treat oneor more infections caused by six, seven, eight, nine, ten, fifteen,twenty or more types of bacteria at the same time.

In certain embodiments, provided herein are methods of treating,preventing and/or managing diseases, disorders, or conditions caused byfungal infection. Examples include, but are not limited to,aspergilliosis, crytococcosis, sporotrichosis, coccidioidomycosis,paracoccidioidomycosis, histoplasmosis, blastomycosis, zygomycosis, andcandidiasis.

In certain embodiments, at least one compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can treat aninfection caused by one type of fungi. In other embodiments, at leastone compound of formula (I) or a pharmaceutically acceptable formthereof, or a pharmaceutical composition comprising at least onecompound of formula (I) or a pharmaceutically acceptable form thereofprovided herein can treat one or more infections caused by two or moretypes of fungi at the same time. In other embodiments, at least onecompound of formula (I) or a pharmaceutically acceptable form thereof,or a pharmaceutical composition comprising at least one compound offormula (I) or a pharmaceutically acceptable form thereof providedherein can treat one or more infections caused by three or more types offungi at the same time. In other embodiments, at least one compound offormula (I) or a pharmaceutically acceptable form thereof, or apharmaceutical composition comprising at least one compound of formula(I) or a pharmaceutically acceptable form thereof provided herein cantreat one or more infections caused by four or more types of fungi atthe same time. In other embodiments, at least one compound of formula(I) or a pharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can treat oneor more infections caused by five or more types of fungi at the sametime. In other embodiments, at least one compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can treat oneor more infections caused by six, seven, eight, nine, ten, fifteen,twenty or more types of fungi at the same time.

In certain embodiments, provided herein are methods of treating,preventing and/or managing diseases, disorders, or conditions caused byparasitic or protozoal infection. Examples of parasitic or protozoaldiseases and disorders include, but are not limited to, diseases,disorders and conditions caused by parasites such as, but not limitedto, P. falcifarium, P. ovale, P. vivax, P. malariae, L. donovari, L.infantum, L. aethiopica, L. major, L. tropica, L. mexicana, L.braziliensis, T. Gondii, B. microti, B. divergens, B. coli, B. hominis,C. parvum, C. cayetanensis, D. fragilis, E. histolytica, I. belli, S.mansonii, S. haematobium, Trypanosoma ssp., Toxoplasma ssp., and O.volvulus. Other diseases, disorders and conditions include, but are notlimited to, those caused by Babesia bovis, Babesia canis, BanesiaGibsoni, Besnoitia darlingi, Cytauxzoon felis, Eimeria ssp., Hammondiassp., T. canis, Cestoda (i.e., tapeworms) and Theileria ssp. Specificdiseases, disorders and conditions include, but are not limited to,malaria, babesiosis, trypanosomiasis, American trypanosomiasis (i.e.,Chagas disease), leishmaniasis, toxoplasmosis, meningoencephalitis,keratitis, amebiasis, giardiasis, cryptosporidiosis, isosporiasis,cyclosporiasis, microsporidiosis, ascariasis, trichuriasis,ancylostomiasis, strongyloidiasis, toxocariasis, trichinosis, lymphaticfilariasis, onchocerciasis, filariasis, schistosomiasis, and dermatitiscaused by animal schistosomes.

In one embodiment, the parasitic or protozoal disease is malaria. Inanother embodiment, the parasitic or protozoal disease is leishmaniasis.In another embodiment, the parasitic or protozoal disease is babesiosis.In another embodiment, the parasitic or protozoal disease istoxoplasmosis. In another embodiment, the parasitic or protozoal diseaseis trypanosomiasis.

In certain embodiments, at least one compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can treat aninfection caused by one type of parasite. In other embodiments, at leastone compound of formula (I) or a pharmaceutically acceptable formthereof, or a pharmaceutical composition comprising at least onecompound of formula (I) or a pharmaceutically acceptable form thereofprovided herein can treat one or more infections caused by two or moretypes of parasite at the same time. In other embodiments, at least onecompound of formula (I) or a pharmaceutically acceptable form thereof,or a pharmaceutical composition comprising at least one compound offormula (I) or a pharmaceutically acceptable form thereof providedherein can treat one or more infections caused by three or more types ofparasite at the same time. In other embodiments, at least one compoundof formula (I) or a pharmaceutically acceptable form thereof, or apharmaceutical composition comprising at least one compound of formula(I) or a pharmaceutically acceptable form thereof provided herein cantreat one or more infections caused by four or more types of parasite atthe same time. In other embodiments, at least one compound of formula(I) or a pharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can treat oneor more infections caused by five or more types of parasite at the sametime. In other embodiments, at least one compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can treat oneor more infections caused by six, seven, eight, nine, ten, fifteen,twenty or more types of parasite at the same time.

In some embodiments, compounds provided herein can treat infection byany combination of viruses, bacteria, fungi and parasites at the sametime. For example, in certain embodiments, compounds provided herein cantreat the infection by one or more viruses and one or more fungi. Inother embodiments, compounds provided herein can treat the infection byone or more viruses and one or more bacteria. In other embodiments,compounds provided herein can treat the infection by one or more fungiand one or more bacteria. In other embodiments, compounds providedherein can treat the infection by one or more viruses and one or moreparasites. In other embodiments, compounds provided herein can treat theinfection by one or more fungi and one or more parasites. In otherembodiments, compounds provided herein can treat the infection by one ormore bacteria and one or more prasites. In other embodiments, compoundsprovided herein can treat the infection by one or more viruses, one ormore fungi and one or more bacteria. In other embodiments, compoundsprovided herein can treat the infection by one or more bacteria, one ormore fungi and one or more parasites. In other embodiments, compoundsprovided herein can treat the infection by one or more viruses, one ormore fungi and one or more parasites. In other embodiments, compoundsprovided herein can treat the infection by one or more viruses, one ormore bacteria and one or more parasites.

Compounds provided herein are inhibitors of FASN. Thus, in certainembodiments, the compounds provided herein can be used to treat and/ormanage other FASN-related disorders, examples of which include, but arenot limited to, diabetes and general wellness of liver such astreatment, prevention and/or management of fatty liver.

In certain embodiments, the compound is an inhibitor of palmitatesynthesis. As used herein “inhibition”, “inhibiting”, “inhibit” and“inhibitor”, and the like, refer to the ability of a compound to reduce,halt or prevent activity of a particular biological process (e.g., FASNactivity, palmitate synthesis) in a cell relative to vehicle.

In other embodiments, provided herein are methods for inhibiting ELOVLin a subject comprising administering to a subject in need thereof atherapeutically effective amount of at least one compound of formula (I)or a pharmaceutically acceptable form thereof.

In another embodiment, provided herein is use of at least one compoundof formula (I) for the treatment of a ELOVL-mediated disorder in asubject.

In another embodiment, provided herein is use of at least one compoundof formula (I) in the manufacture of a medicament. In certainembodiments, the medicament is useful for treating a ELOVL-mediateddisorder.

“ELOVL-mediated disorder” as used herein, refers to a disease, disorderor condition which is treatable by inhibition of ELOVL activity.Typically, ELOVL-mediated disorders are substantially similar to thosemediated by FASN. Thus, ELOVL-mediated disorders include theFASN-mediated disorders described herein above. Examples include, butare not limited to, hyperproliferative disorders, inflammatorydisorders, obesity-related disorders and complications thereof, diabetesand general wellness of liver such as treatment, prevention and/ormanagement of fatty liver.

In one embodiment, the ELOVL-mediated disorder is a hyperproliferativedisorder. In another embodiment, the ELOVL-mediated disorder is aninflammatory disorder. In another embodiment, the ELOVL-mediateddisorder is obesity. In another embodiment, the ELOVL-mediated disorderis diabetes mellitus. In another embodiment, the ELOVL-mediated disorderis fatty liver.

In one embodiment, the ELOVL-mediated disorder is ELOVL6-mediateddisorder.

5. Administration

The compound of formula (I) or a pharmaceutically acceptable formthereof, or a pharmaceutical composition comprising at least onecompound of formula (I) or a pharmaceutically acceptable form thereofcan be administered using any amount and any route of administrationeffective for treatment. The compounds provided herein are typicallyformulated in dosage unit form for ease of administration and uniformityof dosage. It will be understood, however, that the total daily usage ofthe compounds provided herein will be decided by the attending physicianwithin the scope of sound medical judgment. The specific therapeuticallyeffective dose level for any particular subject will depend upon avariety of factors including the disease, disorder, or condition beingtreated and its severity; the activity of the specific compoundemployed; the specific composition employed; the species, age, bodyweight, general health, sex and diet of the subject; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

A therapeutically effective amount of at least one compound of formula(I) or a pharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof disclosed herein can bemeasured by the therapeutic effectiveness of the compound. Compounds offormula (I) can be administered in a dose of about 1 μg/kg to about 200mg/kg daily; such as from about 1 μg/kg to about 150 mg/kg, from about 1mg/kg to about 200 mg/kg, from about 1 μg/kg to about 100 mg/kg, fromabout 1 μg/kg to about 1 mg/kg, from about 50 μg/kg to about 200 mg/kg,from about 10 μg/kg to about 1 mg/kg, from about 10 μg/kg to about 100μg/kg, from about 100 g to about 10 mg/kg, and from about 500 μg/kg toabout 50 mg/kg.

In certain embodiments, a therapeutically effective amount of at leastone compound of formula (I) or a pharmaceutically acceptable formthereof, or a pharmaceutical composition comprising at least onecompound of formula (I) or a pharmaceutically acceptable form thereoffor administration one or more times a day to a 70 kg adult human cancomprise about 0.0001 mg to about 1000 mg of an compound per unit dosageform. It will be appreciated that dose ranges as described hereinprovide guidance for the administration of pharmaceutical compositionsto an adult. The amount to be administered to, for example, a child oran adolescent can be determined by a medical practitioner or personskilled in the art and can be lower or the same as that administered toan adult.

The desired dosage can be delivered three times a day, two times a day,once a day, every other day, every third day, every week, every twoweeks, every three weeks, or every four weeks. In certain embodiments,the desired dosage can be delivered using multiple administrations(e.g., two, three, four, five, six, seven, eight, nine, ten, eleven,twelve, thirteen, fourteen, or more administrations).

In one embodiment, the therapeutically effective amount of a disclosedcompound of formula (I) or a pharmaceutically acceptable form thereof,or a pharmaceutical composition comprising at least one compound offormula (I) or a pharmaceutically acceptable form thereof is sufficientto establish a maximal plasma concentration ranging from about 0.001 μMto about 100 μM, e.g., from about 1 μM to about 20 μM. Preliminary dosesas, for example, determined according to animal tests, and the scalingof dosages for human administration is performed according toart-accepted practices.

The therapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture assays or animalmodels. Levels in plasma can be measured, for example, by highperformance liquid chromatography. The effects of any particular dosagecan be monitored by a suitable bioassay. Examples of dosages are: about0.1×IC₅₀, about 0.5×IC₅₀, about 1×IC₅₀, about 5×IC₅₀, 10×IC₅₀, about50×IC₅₀, and about 100×IC₅₀.

Therapeutically effective dosages achieved in one animal model can beconverted for use in another animal, including humans, using conversionfactors known in the art (see, e.g., Freireich et al., Cancer Chemother.Reports 50(4):219-244 (1966) and Table A for Equivalent Surface AreaDosage Factors).

TABLE A To: Mouse Rat Monkey Dog Human From: (20 g) (150 g) (3.5 kg) (8kg) (60 kg) Mouse 1 1/2 1/4 1/6  1/12 Rat 2 1 1/2 1/4 1/7 Monkey 4 2 13/5 1/3 Dog 6 4 3/5 1 1/2 Human 12 7 3 2 1

In some embodiments, the compound of formula (I) or a pharmaceuticallyacceptable form thereof, or a pharmaceutical composition comprising atleast one compound of formula (I) or a pharmaceutically acceptable formthereof is administered via a variety of routes, including oral,intravenous, intramuscular, intra-arterial, intramedullary, intrathecal,subcutaneous, intraventricular, transdermal, interdermal, rectal,intravaginal, intraperitoneal, topical (as by powders, ointments,creams, and/or drops), mucosal, nasal, bucal, enteral, sublingual; byintratracheal instillation, bronchial instillation, and/or inhalation;and/or as an oral spray, nasal spray, and/or aerosol. Specificallycontemplated routes are systemic intravenous injection, regionaladministration via blood and/or lymph supply, and/or directadministration to an affected site. In general the most appropriateroute of administration will depend upon a variety of factors includingthe nature of the agent (e.g., its stability in the environment of thegastrointestinal tract), the condition of the subject (e.g., whether thesubject is able to tolerate oral administration), etc. At present theoral and/or nasal spray and/or aerosol route is most commonly used todeliver therapeutic agents directly to the lungs and/or respiratorysystem. However, the delivery of the pharmaceutical composition by anyappropriate route, taking into consideration likely advances in thesciences of drug delivery, is also encompassed herein.

It will be also appreciated that at least one compound of formula (I) ora pharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof, as described above and herein,can be administered in combination with one or more additionaltherapeutically active agents.

By “in combination with,” it is not intended to imply that the agentsmust be administered at the same time and/or formulated for deliverytogether, although these methods of delivery are certainly within thescope of this disclosure. The compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof can be administeredconcurrently with, prior to, or subsequent to, one or more otheradditional therapeutically active agents. In general, each agent will beadministered at a dose and/or on a time schedule determined for thatagent. In will further be appreciated that the additionaltherapeutically active agent utilized in this combination can beadministered together in a single composition or administered separatelyin different compositions. The particular combination to employ in aregimen will take into account compatibility of the compound of formula(I) with the additional therapeutically active agent and/or the desiredtherapeutic effect to be achieved.

In some embodiments, additional therapeutically active agents utilizedin combination with at least one compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof will be administered at levelsthat do not exceed the levels at which they are utilized individually.In some embodiments, the levels utilized in combination will be lowerthan those utilized individually.

By a “therapeutically active agent”, “therapeutic agent”, “agent” or“active agent” refers to any substance that is useful for therapy,including prophylactic and therapeutic treatment.

Also encompassed herein is the delivery of the pharmaceuticalcompositions in combination with agents that can improve theirbioavailability, reduce and/or modify their metabolism, inhibit theirexcretion, and/or modify their distribution within the body. It willalso be appreciated that the therapy employed can achieve a desiredeffect for the same disorder (for example, at least one compound offormula (I) or a pharmaceutically acceptable form thereof, or apharmaceutical composition comprising at least one compound of formula(I) or a pharmaceutically acceptable form thereof can be administered incombination with an anti-inflammatory, anti-anxiety and/oranti-depressive agent, etc.), and/or they can achieve different effects(e.g., control of any adverse side-effects).

Exemplary therapeutically active agents include, but are not limited to,anti-cancer agents, antibiotics, anti-obesity drugs, anti-viral agents,anesthetics, anti-coagulants, inhibitors of an enzyme, steroidal agents,anti-inflammatory agents, antihistamine, immunosuppressant agents,anti-neoplastic agents, antigens, vaccines, antibodies, decongestants,sedatives, opioids, pain-relieving agents, analgesics, anti-pyretics,enhancing agents, hormones, prostaglandins, progestational agents,anti-glaucoma agents, ophthalmic agents, anti-cholinergics,anti-depressants, anti-psychotics, hypnotics, tranquilizers,anti-convulsants, muscle relaxants, anti-spasmodics, musclecontractants, channel blockers, miotic agents, anti-secretory agents,anti-thrombotic agents, anticoagulants, anti-cholinergics, 0-adrenergicblocking agents, diuretics, cardiovascular active agents, vasoactiveagents, vasodilating agents, anti-hypertensive agents, angiogenicagents, modulators of cell-extracellular matrix interactions (e.g. cellgrowth inhibitors and anti-adhesion molecules), orinhibitors/intercalators of DNA, RNA, protein-protein interactions,protein-receptor interactions, etc. Active agents include small organicmolecules such as drug compounds (e.g., compounds approved by the Foodand Drugs Administration as provided in the Code of Federal Regulations(CFR)), antibodies, peptides, proteins, carbohydrates, monosaccharides,oligosaccharides, polysaccharides, nucleoproteins, mucoproteins,lipoproteins, synthetic polypeptides or proteins, small molecules linkedto proteins, glycoproteins, steroids, nucleic acids, DNAs, RNAs,nucleotides, nucleosides, oligonucleotides, antisense oligonucleotides,lipids, hormones, antibodies, vitamins and cells, and combinationsthereof.

In certain embodiments, the therapeutically active agent is ananti-cancer agent. Exemplary anti-cancer agents, include, but are notlimited to, radiation therapy, interferon (e.g., interferon α,interferon γ), antibodies (e.g., HERCEPTIN (trastuzumab), T-DM1, AVASTIN(bevacizumab), ERBITUX (cetuximab), VECTIBIX (panitumumab), RITUXAN(rituximab) BEXXAR (tositumomab)), anti-estrogens (e.g., tamoxifen,raloxifene, and megestrol), LHRH agonists (e.g., goscrclin andleuprolide), anti-androgens (e.g., flutamide and bicalutamide),photodynamic therapies (e.g., vertoporfin (BPD-MA), phthalocyanine,photosensitizer Pc4, and demethoxy-hypocrellin A (2BA-2-DMHA)), nitrogenmustards (e.g., cyclophosphamide, ifosfamide, trofosfamide,chlorambucil, estramustine, and melphalan), nitrosoureas (e.g.,carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g.,busulfan and treosulfan), triazenes (e.g., dacarbazine, temozolomide),platinum containing compounds (e.g., cisplatin, carboplatin,oxaliplatin), vinca alkaloids (e.g., vincristine, vinblastine,vindesine, and vinorelbine), taxoids (e.g., paclitaxel, albumin-boundpaclitaxel (ABRAXANE), nab-paclitaxel, docetaxel, taxol),epipodophyllins (e.g., etoposide, etoposide phosphate, teniposide,topotecan, 9-aminocamptothecin, camptoirinotecan, irinotecan, crisnatol,mytomycin C), anti-metabolites, DHFR inhibitors (e.g., methotrexate,dichloromethotrexate, trimetrexate, edatrexate), IMP dehydrogenaseInhibitors (e.g., mycophenolic acid, tiazofurin, ribavirin, and EICAR),ribonuclotide reductase inhibitors (e.g., hydroxyurea and deferoxamine),uracil analogs (e.g., 5-fluorouracil (5-FU), floxuridine, doxifluridine,ratitrexed, tegafur-uracil, capecitabine), cytosine analogs (e.g.,cytarabine (ara C), cytosine arabinoside, and fludarabine), purineanalogs (e.g., mercaptopurine and Thioguanine), Vitamin D3 analogs(e.g., EB 1089, CB 1093, and KH 1060), isoprenylation inhibitors (e.g.,lovastatin), dopaminergic neurotoxins (e.g., 1-methyl-4-phenylpyridiniumion), cell cycle inhibitors (e.g., staurosporine), actinomycin (e.g.,actinomycin D, dactinomycin), bleomycin (e.g., bleomycin A2, bleomycinB2, peplomycin), anthracycline (e.g., daunorubicin, doxorubicin,pegylated liposomal doxorubicin, idarubicin, epirubicin, pirarubicin,zorubicin, mitoxantrone), MDR inhibitors (e.g., verapamil), Ca2+ ATPaseinhibitors (e.g., thapsigargin), imatinib, thalidomide, lenalidomide,tyrosine kinase inhibitors tyrosine kinase inhibitors (e.g., axitinib(AG013736), bosutinib (SKI-606), cediranib (RECENTIN™, AZD2171),dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®), gefitinib(IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib (TYKERB®,TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272), nilotinib(TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®, SU11248),toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474), vatalanib (PTK787,PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab (AVASTIN®), rituximab(RITUXAN®), cetuximab (ERBITUX®), panitumumab (VECTIBIX®), ranibizumab(Lucentis®), nilotinib (TASIGNA®), sorafenib (NEXAVAR®), everolimus(AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumab ozogamicin (MYLOTARG®),temsirolimus (TORISEL®), ENMD-2076, PCI-32765, AC220, dovitinib lactate(TK1258, CHIR-258), BIBW 2992 (TOVOK™), SGX523, PF-04217903,PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF 1120(VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154,CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/orXL228), proteasome inhibitors (e.g., bortezomib (VELCADE)), mTORinhibitors (e.g., rapamycin, temsirolimus (CCI-779), everolimus(RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235(Novartis), BGT226 (Norvartis), XL765 (Sanofi Aventis), PF-4691502(Pfizer), GDC0980 (Genetech), SF1126 (Semafoe) and OSI-027 (OSI)),oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed,cyclophosphamide, dacarbazine, procarbizine, prednisolone,dexamethasone, campathecin, plicamycin, asparaginase, aminopterin,methopterin, porfiromycin, melphalan, leurosidine, leurosine,chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin,aminopterin, and hexamethyl melamine.

Exemplary combinations of therapeutically active agents useful for thetreatment of cancer (a.k.a. an “anti-cancer treatment regimen”) whichcan be used in combination with at least one compound of formula (I) ora pharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof includes, but is not limitedto:

ABVD Adriamycin (doxorubicin), bleomycin, vinblastine, dacarbazine ACAdriamycin (doxorubicin), cyclophosphamide BEACOPP Bleomycin, etoposide,Adriamycin (doxorubicin), cyclophosphamide, Oncovin (vincristine),procarbazine, prednisone BEP Bleomycin, etoposide, platinum agent(cisplatin) CA Cyclophosphamide, Adriamycin (doxorubicin) (same as AC)CAF Cyclophosphamide, Adriamycin (doxorubicin), fluorouracil (5-FU) CAVCyclophosphamide, Adriamycin (doxorubicin), vincristine CBVCyclophosphamide, BCNU (carmustine), VP-16 (etoposide) ChlVPP/EVAChlorambucil, vincristine (Oncovin), procarbazine, prednisone,etoposide, vinblastine, Adriamycin (doxorubicin) CHOP Cyclophosphamide,hydroxydoxorubicin (doxorubicin), vincristine (Oncovin), prednisoneCHOP-R or R-CHOP CHOP + rituximab COP or CVP Cyclophosphamide, Oncovin(vincristine), prednisone CMF Cyclophosphamide, methotrexate,fluorouracil (5-FU) COPP Cyclophosphamide, Oncovin (vincristine),procarbazine, prednisone EC Epirubicin, cyclophosphamide ECF Epirubicin,cisplatin, fluorouracil (5-FU) EP Etoposide, platinum agent (cisplatin)EPOCH Etoposide, prednisone, Oncovin, cyclophosphamide, andhydroxydaunorubicin FEC Fluorouracil (5-FU), epirubicin,cyclophosphamide FL (Also known as Mayo) Fluorouracil (5-FU), leucovorin(folinic acid) FOLFOX Fluorouracil (5-FU), leucovorin (folinic acid),oxaliplatin FOLFIRI Fluorouracil (5-FU), leucovorin (folinic acid),irinotecan ICE Ifosfamide, carboplatin, etoposide (VP-16) ICE-R ICE +rituximab m-BACOD Methotrexate, bleomycin, Adriamycin (doxorubicin),cyclophosphamide, Oncovin (vincristine), dexamethasone MACOP-BMethotrexate, leucovorin (folinic acid), Adriamycin (doxorubicin),cyclophosphamide, Oncovin (vincristine), prednisone, bleomycin MOPPMechlorethamine, Oncovin (vincristine), procarbazine, prednisone PCVProcarbazine, CCNU (lomustine), vincristine ProMACE-MOPP Methotrexate,Adriamycin (doxorubicin), cyclophosphamide, etoposide + MOPPProMACE-CytaBOM Prednisone, doxorubicin (adriamycin), cyclophosphamide,etoposide, cytarabine, bleomycin, Oncovin (vincristine), methotrexate,leucovorin R-FCM Rituximab, fludarabine, cyclophosphamide, mitoxantroneStanford V Doxorubicin, mechlorethamine, bleomycin, vinblastine,vincristine, etoposide, prednisone Thal/Dex Thalidomide, dexamethasoneTIP Paclitaxel, ifosfamide, platinum agent cisplatin VAC Vincristine,Actinomycin, Cyclophosphamide VAD Vincristine, Adriamycin (doxorubicin),dexamethasone VAPEC-B Vincristine, Adriamycin (doxorubicin), prednisone,etoposide, cyclophosphamide, bleomycin VIP Etoposide, ifosfamide,platinum agent cisplatin

In other embodiments, the therapeutically effective agent is ananti-vial agent. Exemplary anti-viral agents include, but are notlimited to, Abacavir, Aciclovir, Acyclovir, Adefovir, Amantadine,Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla, BI201335,Boceprevir, BMS-858 (see, e.g., Gao et al., Nature, 465(6): 96-102(2010)), BMS-790052 ((see, e.g., Gao et al., Nature, 465(6): 96-102(2010)), Cidofovir, Combivir, Danoprivir (ITMN-191; RG-7227), Darunavir,Delavirdine, Didanosine, Docosanol, Edoxudine, EI-1 to EI-12 (see, e.g.,Baldick et al., PLoS Pathogens, 6(9)e1001086: 1-14 (2010)),Elvitegravir, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir,Etravirine, Famciclovir, Fosamprenavir, Foscarnet, Fosfonet,Ganciclovir, GSK-572, Ibacitabine, Imunovir, Idoxuridine, Imiquimod,Indinavir, Inosine, Interferon (e.g., Interferon type III, Interferontype II, Interferon type I, Peginterferon alfa-2a, Peginterferonalpha-2b, standard interferon alfa-2a, standard interferon alfa-2b,consensus interferon, interferon alfacon-1, ALBUFERON, omega interferon,interferon gamma-1b, lymphoblastoid interferon tau), Lamivudine,Lopinavir, Loviride, Maraviroc, Moroxydine, Methisazon, MK-2048,Nelfinavir, Nevirapine, Nexavir, Oseltamivir (Tamiflu), Penciclovir,Peramivir, Pleconaril, Podophyllotoxin, Raltegravir, Ribavirin,Rimantadine, Ritonavir, Pyramidine, Saquinavir, Stavudine, Tenofovir(e.g., Tenofovir disoproxil), Telaprivir, Tipranavir, Trifluridine,Trizivir, Tromantadine, Truvada, Valaciclovir (Valtrex), Valganciclovir,vaccines (e.g., VZV vaccines such as Varivax and Zostavax), Vicriviroc,Vidarabine, Viramidine, Zalcitabine, Zanamivir (Relenza), Zidovudine,and other small moelcule anti-viral agents described, for example, inHerker et al., Nature Medicine, Advance Online Publicationdoi:10.1038/nm2238: 1-4 (Oct. 10, 2010), and combinations thereof.

Examples of additional anti-viral agents include, but are not limitedto, interleukin 2, interleukin 6, inteleukin 12, a compound thatenhances the development of a type 1 helper T cell response, interferingRNA, anti-sense RNA, Imiqimod, an inosine 5′-phosphate dehydrogenaseinhibitor, amantadine and rimantadine.

Other examples include, but are not limited to, those described in WO2009/023059, the entirety of which is incorporated herein by reference.

In one embodiment, the anti-viral agent is interferon. In anotherembodiment, the anti-viral agent is telaprivir. In one embodiment,combinations of two or more anti-viral agents are used in furthercombination with a compound provided herein.

In certain embodiments, the anti-viral agent is a protease inhibitor.Exemplary protease inhibitors include, but are not limited to,Saquinavir, Ritonavir, Indinavir, Nelfinavir, Amprenavir, Lopinavir,Atazanavir, Fosamprenavir, Tipranavir and Darunavir.

In certain embodiments, the anti-viral agent is an integrase inhibitor.Exemplary integrase inhibitors include, but are not limited to,Raltegravir, Elvitegravir and MK-2048, GSK-572.

In certain embodiments, the anti-viral agent is a reverse transcriptaseinhibitor (e.g., a nucleoside analog reverse trascriptase inhibitor(NRTI), a nucleotide analog reverse trascriptase inhibitor (NtRTI), anon-nucleoside reverse transcripase inhibitor (NNRTI)).

Exemplary nucleoside analog reverse trascriptase inhibitors (NRTIs)include, but are not limited to, Zidovudine, Didanosine, Zalcitabine,Stavudine, Lamivudine, Abacavir, Emtricitabine, Entecavir and Aciclovir(partial nucleoside structure).

Exemplary nucleotide analog reverse trascriptase inhibitors (NtRTIs)include, but are not limited to, Tenofovir and Adefovir.

Exemplary non-nucleoside reverse transcripase inhibitors (NNRTIs)include, but are not limited to, Efavirenz, Nevirapine, Delavirdine andEtravirine. In certain embodiments, the compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein and/or theanti-viral agent is further used in combination with an enhancing agent.An “enhancing agent”, used in this context, is an agent which, when usedin combination with a compound provided herein and/or an anti-viralagent, improves treatment, prevention or management of the microbialinfection relative to treatment with the compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein and/or ananti-viral agent without the enhancing agent. Exemplary enhancing agentsinclude, but are not limited to, chloroquine, a quinoline antimalarial,grapefruit juice, hydroxyurea, leflunomide, myucophenolic acid,resveratrol and Ritonavir.

In one embodiment, the anti-viral agent is an anti-viral agent describedin U.S. Pub. No. 2011/0064698, which is incorporated herein by referencein its entirety. Exemplary anti-viral agents include, but are notlimited to, IP-501, Merimebodib VX-497, IDN-6556, XTL-002, HCV/MF59,CIVACIR, ZADAXIN, CEPLENE, VX 950/LY 570310, ISIS 14803, JTK 003,Tarvacin, HCV-796, CH-6, ANA971, ANA245, CPG 10101, Rituximab, NM 283,HepX™-C, IC41, Medusa interferon, E-1, multiferon, BILN 2061, TMC435350,Telaprevir, Boceprevir, ACH-1625, ABT-450, BI-201335, PHX-1766, VX-500,MK-7009, R7227, Narlaprevir, Alinia, ABT-072, ABT-333, Filibuvir,VCH-916, R7128, IDX 184, R7128, R1626, MK-3281, PSI-7851, ANA 598,BI-207127, GS9190, VCH-759, Clemizole, A-832, BMS-790052, ITX 5061,GS-9450, ANA773, CYT 107, SPC3649, Debio 25, SCY-635 and a combinationthereof.

Other examples include, but are not limited to, AZD-7295, BI207127,BIT225, BM824383, BMS65032, BMS791325, GS-9256, IDX 375, INX-189,PPI-461, PSI-938, PSI-7977, TMC435, TMC649128, VX-222, VX-759, VX-916and a combination thereof. These agents are currently in various stagesof clinical trials and information is readily available to those in theart.

In one embodiment, provided herein is a method of treating, preventingand/or managing hepatitis C virus (HCV) infection comprisingadministering a therapeutically or prophylactically effective amount ofat least one compound of formula (I) or a pharmaceutically acceptableform thereof, or a pharmaceutical composition comprising at least onecompound of formula (I) or a pharmaceutically acceptable form thereofprovided herein in combination with one or more other therapeutic agentsprovided herein.

Examples of such therapeutic agents include compounds having anti-HCVactivity, for example, by inhibiting the function of a target such as,but not limited to, HCV metalloprotease, HCV serine protease, HCVpolymerase, HCV helicase, SCV NS4B protein, HCV entry, HCV assembly, HCVegress, HCV NS5A protein and IMPDH.

In other embodiments, at least one compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can be used incombination with at least one additional therapeutic agent havinganti-HCV activity, including, but not limited to, Alinia (Nitazoxanide),Bavituximab, Belerofon, Chronvac-C, Civacir, Clemizole, Fluvastatin,Glycoferon, Hepavaxx C, HuMax-HepC, Lenocta (sodium stibogluconate SSG),Locteron, peginterferon, Ribavirin, Suvus, Telaprevir (VX-950),Zadaxin-thymalfasin, ZALBIN (Albuferon albinterferon alfa-2b), A-837093,ABT-072, ABT-333, ABT-450, ACH-1095, ACH-1625, ACH-2684, ACH-2928, AN025-1, ANA598, ANA773, ATI-0810 (formerly PG301029), AVL-181, AVL-192,AZD7295, BI 201335, BI 207127, BIT225, BMS-650032, BMS-790052,BMS-791325, BMS-824393, CB5300, CB-183872 (formerly IB657), CF102,CSL123, CTS-1027, CYT107, Debio 025, ECH18, EDP-239, GEA007.1, GI 5005,GNI-103, GNI-104, GS 9190, GS 9256, GSK625433, IC41, ID-12, IDX184,IDX320, IDX375, IMO-2125, IMMU 105, ITMN-191 R7227 (R05190591), ITX2155,ITX4520, ITX5061NS5A inhibitors, JKB-122, KPE02001003, KPE00001113,MBL-HCV1, MDX-1106 (ONO-4538), Mito-Q, MK-0608, MX3235 Celgosivir,NOV-205, PF-868554, PF-4878691, PHX1766, PYN17, PYN18, PPI-461,PPI-1301, PRO-206, PSI-7977, PSI-938INX08189, R7128 (R05024048), REP 9C,RG7348, SCV-07, SCY-635, SD-101, SIRNA-034, SP-30, SPC3649, TG4040,TT033, VCH-759, VX-222, VX-500, VX-813, and VX-985.

In one embodiment, the other therapeutic agent is an interferon. In oneembodiment, the interferon is Interferon type III, Interferon type II,Interferon type I, Peginterferon alfa-2a, Peginterferon alpha-2b,standard interferon alfa-2a, standard interferon alfa-2b, consensusinterferon, interferon alfacon-1, ALBUFERON, omega interferon,interferon gamma-1b, lymphoblastoid interferon tau or a combinationthereof. In another embodiment, the interferon is interferon alfa-2a,interferon alfa-2b, peginterferon alfa-2a, peginterferon alpha-2b,consensus interferon or lymphoblastoid interferon tau.

In another embodiment, the other therapeutic agent is ribavirin.

In another embodiment, at least one compound of formula (I) or apharmaceutically acceptable form thereof, or a pharmaceuticalcomposition comprising at least one compound of formula (I) or apharmaceutically acceptable form thereof provided herein can be used incombination with ribavirin and an interferon. In one embodiment, theinterferon is Interferon type III, Interferon type II, Interferon typeI, Peginterferon alfa-2a, Peginterferon alpha-2b, standard interferonalfa-2a, standard interferon alfa-2b, consensus interferon, interferonalfacon-1, ALBUFERON, omega interferon, interferon gamma-1b,lymphoblastoid interferon tau or a combination thereof. In anotherembodiment, the interferon is interferon alfa-2a, interferon alfa-2b,peginterferon alfa-2a, peginterferon alpha-2b, consensus interferon orlymphoblastoid interferon tau.

6. Anti-Viral Assays

Anti-viral assays used to screen compounds having efficacy for aspecific virus are well-known in the art and described, for example, inWO 2009/023059, the entirety of which is incorporated herein byreference. Exemplary anti-viral assays are provided herein below.

6.1 Herpes Simplex Virus (HSV)

Mouse models of herpes simplex virus type 1 or type 2 (HSV-1 or HSV-2)can be employed to assess the anti-viral activity of test compounds invivo. BALB/c mice are commonly used, but other suitable mouse strainsthat are susceptible can also be used. Mice are inoculated by variousroutes with an appropriate multiplicity of infection of HSV, followed byadministration of test compounds and placebo. For i.p. inoculation,HSV-1 replicates in the gut, liver, and spleen and spreads to the CNS.For i.n. inoculation, HSV-1 replicates in the nasaopharynx and spreadsto the CNS. Any appropriate route of administration (e.g., oral,topical, systemic and nasal), frequency and dose of administration canbe tested to determine the optimal dosages and treatment regimens usingtest compounds, optionally in combination with other therapies.

In a mouse model of HSV-2 genital disease, intravaginal inoculation offemale Swiss Webster mice with HSV-1 or HSV-2 is carried out, andvaginal swabs are obtained to evaluate the effect of therapy on viralreplication. (See, e.g., Crute et al., Nature Medicine, 2002,8:386-391). For example, viral titers by plaque assays are determinedfrom the vaginal swabs. A mouse model of HSV-1 using SKH-1 mice, astrain of immunocompetent hairless mice, to study cutaneous lesions isalso described in the art. (See, e.g., Crute et al., Nature Medicine,2002, 8:386-391 and Bolger et al., Antiviral Res., 1997, 35:157-165).Guinea pig models of HSV have also been described. (See, e.g., Chen etal., Virol. J., 2004 Nov. 23, 1:11). Statistical analysis is typicallycarried out to calculate significance of the anti-viral activity.

6.2 Human Cytomegalovirus (HCMV)

Since HCMV does not generally infect laboratory animals, mouse models ofinfection with murine CMV (MCMV) can be used to assay anti-viralactivity of test compounds in vivo. For example, a MCMV mouse model withBALB/c mice can be used to assay the anti-viral activities of testcompounds in vivo when administered to infected mice, which isdescribed, for example, in Kern et al., Antimicrob. Agents Chemother.,2004, 48:4745-4753. Tissue homogenates isolated from infected micetreated or untreated with test compounds are tested using standardplaque assays with mouse embryonic fibroblasts (MEFs). Statisticalanalysis is then typically carried out to calculate significance of theanti-viral activity.

Alternatively, human tissue (i.e., retinal tissue or fetal thymus andliver tissue) is implanted into SCID mice, and the mice are subsequentlyinfected with HCMV, preferably at the site of the tissue graft. (See,e.g., Kern et al., Antimicrob. Agents Chemother., 2004, 48:4745-4753).The pfu of HCMV used for inoculation can vary depending on theexperiment and virus strain. Any appropriate routes of administration(e.g., oral, topical, systemic and nasal), frequency and dose ofadministration can be tested to determine the optimal dosages andtreatment regimens using test compounds, optionally in combination withother therapies. Implant tissue homogenates isolated from infected micetreated or untreated with test compounds at various time points aretested using standard plaque assays with human foreskin fibroblasts(HFFs). Statistical analysis is then typically carried out to calculatesignificance of the anti-viral activity.

Guinea pig models of CMV to study anti-viral agents have also beendescribed, for example, in Bourne et al., Antiviral Res., 2000,47:103-109; Bravo et al., Antiviral Res., 2003, 60:41-49; and Bravo etal, J. Infectious Diseases, 2006, 193:591-597.

6.3 Influenza Virus

Animal models, such as ferret, mouse and chicken, developed for use totest anti-viral agents against influenza virus have been described, forexample, in Sidwell et al., Antiviral Res., 2000, 48: 1-16 and McCauleyet al., Antiviral Res., 1995, 27: 179-186. For mouse models ofinfluenza, non-limiting examples of parameters that can be used to assayanti-viral activity of test compounds administered to theinfluenza-infected mice include pneumonia-associated death, serumal-acid glycoprotein increase, animal weight, lung virus assayed byhemagglutinin, lung virus assayed by plaque assays, andhistopathological change in the lung. Statistical analysis is typicallycarried out to calculate significance of the anti-viral activity.

Nasal turbinates and trachea can be examined for epithelial changes andsubepithelial inflammation. The lungs can be examined for bronchiolarepithelial changes and peribronchiolar inflammation in large, medium,and small or terminal bronchioles. The alveoli are also evaluated forinflammatory changes. The medium bronchioles are graded on a scale of 0to 3+ as follows: 0 (normal: lined by medium to tall columnar epithelialcells with ciliated apical borders and basal pseudostratified nuclei;minimal inflammation); 1+(epithelial layer columnar and even in outlinewith only slightly increased proliferation; cilia still visible on manycells); 2+(prominent changes in the epithelial layer ranging fromattenuation to marked proliferation; cells disorganized and layeroutline irregular at the luminal border); and 3+(epithelial layermarkedly disrupted and disorganized with necrotic cells visible in thelumen; some bronchioles attenuated and others in marked reactiveproliferation).

The trachea is graded on a scale of 0 to 2.5+ as follows: 0 (normal:Lined by medium to tall columnar epithelial cells with ciliated apicalborder, nuclei basal and pseudostratified. Cytoplasm evident betweenapical border and nucleus. Occasional small focus with squamous cells);1+(focal squamous metaplasia of the epithelial layer); 2+(diffusesquamous metaplasia of much of the epithelial layer, cilia may beevident focally); and 2.5+(diffuse squamous metaplasia with very fewcilia evident).

Virus immunohistochemistry is performed using a viral-specificmonoclonal antibody (e.g., NP-, N- or HN-specific monoclonalantibodies). Staining is graded 0 to 3+ as follows: 0 (no infectedcells); 0.5+(few infected cells); 1+(few infected cells, as widelyseparated individual cells); 1.5+(few infected cells, as widelyseparated singles and in small clusters); 2+(moderate numbers ofinfected cells, usually affecting clusters of adjacent cells in portionsof the epithelial layer lining bronchioles, or in small sublobular fociin alveoli); and 3+(numerous infected cells, affecting most of theepithelial layer in bronchioles, or widespread in large sublobular fociin alveoli).

6.4 Hepatitis Type B Virus (HBV)

A HBV transgenic mouse model, lineage 1.3.46 (official designation,Tg[HBV 1.3 genome] Chi46) has been described previously and can be usedto test the in vivo anti-viral activities of test compounds as well asthe dosing and administration regimen. (See, e.g., Cavanaugh et al., J.Virol., 1997, 71:3236-3243; and Guidotti et al., J. Virol., 1995,69:6158-6169). In these HBV transgenic mice, a high level of viralreplication occurs in liver parenchymal cells and in the proximalconvoluted tubules in the kidneys of these transgenic mice at levelscomparable to those observed in the infected liver of patients withchronic HBV hepatitis. HBV transgenic mice that have been matched forage (i.e., 6-10 weeks), sex (i.e., male), and levels of hepatitis Bsurface antigen (HBsAg) in serum can be treated with test compounds orplacebo followed by anti-viral activity analysis to assess the activityof test compounds. Non-limiting examples of assays that can be performedon these mice treated and untreated with test compounds include southernanalysis to measure HBV DNA in the liver, quantitative reversetranscriptase PCR (qRT-PCR) to measure HBV RNA in the liver,immunoassays to measure hepatitis e antigen (HBeAg) and HBV surfaceantigen (HBsAg) in the serum, immunohistochemistry to measure HBVantigens in the liver, and quantitative PCR (qPCR) to measure serum HBVDNA. Gross and microscopic pathological examinations can be performed asneeded.

6.5 Human Immunodeficiency Virus (HIV)

The safety and efficacy of test compounds against HIV can be assessed invivo with established animal models well-known in the art. For example,a Trimera mouse model of HIV-1 infection has been developed byreconstituting irradiated normal BALB/c mice with murine SCID bonemarrow and engrafted human peripheral blood mononuclear cells. (SeeAyash-Rashkovsky et al., FASEB J., 2005, 19:1149-1151). These mice areinjected intraperitoneally with T- and M-tropic HIV-1 laboratorystrains. After HIV infection, rapid loss of human CD4.sup.+ T cells,decrease in CD4/CD8 ratio, and increased T cell activation can beobserved. A test compound can be administered to these mice and standardassays known in the art can be used to determine the viral replicationcapacity in animals treated or untreated with the compound. Non-limitingexamples of such assays include the COBAS AMPLICOR™ RT-PCR assay (RocheDiagnostics, Branchberg, N.J.) to determine plasma viral load (HIV-1 RNAcopies/ml); active HIV-1 virus replication assay where human lymphocytesrecovered from infected Trimera mice were cocultured with target T cells(MT-2 cells) and HIV-dependent syncytia formation was examined; andhuman lymphocytes recovered from infected Trimera mice were coculturedwith cMAGI indicator cells, where HIV-1 LTR driven trans-activation of3-galactosidase was measured. Levels of anti-HIV-1 antibodies producedin these mice can also be measured by ELISA. Other established mousemodels described in the art can also be used to test the anti-viralactivity of test compounds in vivo. (See, e.g., Mosier et al., Semin.Immunol., 1996, 8:255-262; Mosier et al., Hosp. Pract. (Off Ed), 1996,31:41-48, 53-55, 59-60; Bonyhadi et al., Mol. Med. Today, 1997,3:246-253; Jolicoeur et al., Leukemia, 1999, 13:S78-S80; Browning etal., Proc. Natl. Acad. Sci. USA, 1997, 94:14637-14641; and Sawada etal., J. Exp. Med., 1998, 187:1439-1449). A simian immunodeficiency virus(SIV) nonhuman primate model has also been described, for example, inSchito et al., Curr. HIVRes., 2006, 4:379-386.

6.6 In Vitro Screening Assays

6.6.1 General Procedures for Assays for Herpes Viruses

To quickly screen out samples that do not have activity against any ofthe herpes viruses, or are too toxic to evaluate, an inexpensive, rapidassay such as a CPE-inhibition assay that is semi-automated is commonlyused initially to screen out the negatives. Typically, all screeningassays are conducted in low passage human cells, and each assay systemcontains a positive control (ACV, GCV, CDV) and a negative control(AZT). Efficacy is demonstrated by at least two different assay systemsthat detect functional biologic activity and should be confirmed usinglow passaged clinical isolates and drug resistant mutants wheneveravailable. In the case of EBV, efficacy against EBV is confirmed using ahybridization assay that quantifies DNA synthesis. Toxicity isdetermined using both resting and proliferating human fibroblast cellsand proliferating lymphoblastic cells, and for selected compounds,toxicity in human myeloid and erythroid progenitor cells is assessed.

6.6.1.1 HSV-1, HSV-2, CMV and VZV

All the screening assay systems utilized are selected to show specificinhibition of a biologic function, i.e., cytopathic effect (CPE) insusceptible human cells. In the CPE-inhibition assay, a test compound isadded 1 hour prior to infection so the assay system will have maximumsensitivity and detect inhibitors of early replicative steps, such asadsorption or penetration, as well as later events. To rule outnon-specific inhibition of virus binding to cells, all compounds thatshow reasonable activity in the CPE assay are confirmed using aclassical plaque reduction assay in which the compound is added 1 hourafter infection. In the case where a compound blocks attachment, apositive result will appear in the CPE assay, but may be negative byplaque assay. In this case, the plaque assay is repeated with compoundbeing added prior to viral infection. These assay systems also can bemanipulated by increasing the pretreatment time in order to demonstrateanti-viral activity with oligodeoxynucleotides and/or peptides and bydelaying addition of drug after infection. Information regarding whichstep in the virus life cycle is inhibited (i.e., early vs. latefunctions) can be gained.

In all the assays used for primary screening, a minimum of six compoundconcentrations is contemplated to cover a range of, e.g., 100 μg/ml to0.03 μg/ml, in 5-fold increments to determine efficacy. Dose responsecurves are obtained from these data. The dose that inhibits viralreplication by 50% (effective concentration 50; EC₅₀) is typicallycalculated using a computer software program, for example, MacSynergy IIby M. N. Prichard, K. R. Asaltine, and C. Shipman, Jr., University ofMichigan, Ann Arbor, Mich.

The same compound concentrations used to determine efficacy are alsoused on uninfected cells in each assay to determine toxicity of eachexperimental compound. The compound concentration that is cytotoxic tocells as determined by their failure to take up a vital stain, neutralred (cytotoxic concentration 50; CC₅₀), is determined as describedabove.

In some embodiments, compounds to treat herpes virus infections are forsystemic diseases, such as neonatal herpes, CMV, and disseminated VZV,and may need to be given parenterally. Therefore, the toxicity of testcompounds on dividing cells is determined at a very early stage oftesting. In this regard, a cell proliferation assay using HFF cells canbe a very sensitive assay for detecting compound toxicity to dividingcells, and the compound concentration that inhibits cell growth by 50%(IC₅₀) can be calculated as described above. In comparison with fourhuman diploid cell lines and vero cells, HFF cells are the mostsensitive and predictive of toxicity for bone marrow cells.

To determine if each compound has sufficient anti-viral activity thatexceeds its level of toxicity, a selectivity index (SI) is calculatedaccording to CC₅₀/EC₅₀. This index, also referred to as a therapeuticindex, is used to determine if a compound warrants further study.Typically, a compound that had an SI of 10 or greater is evaluated inadditional assay systems.

For HSV-1 and HSV-2, compounds that show activity in the CPE-inhibitionassay are confirmed using the plaque reduction assay. Susceptibility ofadditional virus strains, including both lab passaged and clinicalisolates, is determined for selected compounds. A battery of ACVresistant HSV strains can be also utilized. For CMV, compounds that showactivity in the CPE-inhibition assay are confirmed using the plaquereduction assay in HFF cells. A variety of laboratory, clinical, and GCVresistant isolates are also available for testing. For VZV, compoundswith activity in a CPE assay are evaluated further in a plaque reductionassay.

6.6.1.2 Epstein-Barr Virus (EBV)

The initial system to be used to determine anti-viral activity againstEBV can be VCA production in Daudi cells using an ELISA assay. Sixconcentrations of drug covering a range of, e.g., 50 μg/ml to 0.03 μg/mlare utilized. Using the results obtained from untreated and drug treatedcells, an EC₅₀ can be calculated. Selected compounds that have goodactivity against EBV VCA production without toxicity are tested fortheir ability to inhibit EBV DNA synthesis.

In each assay system utilized, drug treatment of uninfected cells isincorporated to obtain as much toxicity data as possible. In someembodiments, for calculation of the SI, the data on toxicity is at leastas reliable as the results for efficacy. An example of a toxicity assayis a colormetric method using MTS.

All compounds that have an SI of, for example, greater than 10 in thescreening assay are confirmed in a hybridization assay that measures theamount of EBV DNA produced by P3HR-1 infected cells. A wide range ofcompound concentrations can be utilized so an accurate EC₅₀ can becalculated. Uninfected control cells treated with compound are alsoutilized as another measure of drug toxicity. In some cases, it ispossible that results obtained using assays for VCA production and DNAsynthesis may not correlate since the two events may be independent.

6.6.1.3 Human Herpes Virus HHV-6 and HHV-8

Cord Blood Lymphocytes (CBL) and the Human T cell lymphoblastoid lines,HSB-2 and SupT-1, are used in screening assays for HHV-6. CBL areisolated from fresh heparinized umbilical cord blood and are infectedwith the Z29 strain of HHV-6. The body cavity based B-cell lymphoma cellline, BCBL-1, are used for screening against HHV-8.

There are two variants of HHV-6 known as type A variants or type Bvariants. The HHV-6 type A variant is, for example, the GS strain whichis propagated in HSB-2 or SupT-1 cells. The HHV-6 type B variant is, forexample, Z29 (ATCC, Rockville, Md.) which is grown as a stock in CBL.The HHV-8 is propagated in a latent state in the BCBL-1 cell line. Lyticgrowth of the HHV-8 can be induced by the addition of the phorbol ester,TPA.

Six concentrations of each drug ranging from, e.g., 100 μg/ml to 0.03μg/ml drug are tested to obtain the EC₅₀, EC₉₀, CC₅₀, and IC₅₀ values.The initial assay for HHV-6 is a flow cytometric analysis of HHV-6antigens in either HSB-2 cells (HHV-6A), CBL (HHV-6B), or SupT-1 (6A or6B). For HHV-8, yytic infection of virus in BCBL-1 cells will be carriedout as described above. The initial assay for HHV-8 is a flow cytometricanalysis of HHV-8 antigens in BCBL-1 cells. As with the other herpesvirus assays, these assays contain the positive (infected and untreatedcells) and negative (uninfected or uninduced and compound treated cells)controls needed for effective analysis and cytotoxicity determinations.

6.6.2 In Vitro Laboratory Procedures for Assays for Herpes Viruses

6.6.2.1 Efficacy Screening for HSV-1, HSV-2, CMV and VZV

Preparation of Human Foreskin Fibroblast Cells:

Newborn human foreskins can be obtained from the University of AlabamaSchool of Medicine (UAB) or Brookwood Hospital, Birmingham, Ala., assoon as possible after circumcisions are performed and placed in minimalessential medium (MEM) containing vancomycin, fungizone, penicillin, andgentamicin, at the usual concentrations, for four hours at roomtemperature. The medium is then removed, the foreskin minced into smallpieces and washed repeatedly until red cells are no longer present. Thetissue is then trypsinized using trypsin at 0.25% with continuousstirring for 15 minutes at 37° C. in a CO₂ incubator. At the end of each15 minute period, the tissue is allowed to settle to the bottom of theflask. The supernatant containing cells is poured through sterilecheesecloth into a flask containing MEM and 10% fetal bovine serum(FBS). The flask containing the medium is kept on ice throughout thetrypsinizing procedure. After each decanting of cells, the cheese clothis washed with a small amount of MEM containing serum. Fresh trypsin isadded each time to the foreskin pieces and the procedure repeated untilno more cells become available. The cell-containing medium is thencentrifuged at 1000 RPM at 4° C. for 10 minutes. The supernatant liquidis discarded and the cells are resuspended in a small amount of MEM with10% FBS. The cells are counted using a Coulter Counter and then placedin an appropriate number of 25 cm² tissue culture flasks. As cellsbecome confluent and need trypsinization, they are gradually expandedinto 175 cm² flasks. The cells are maintained on vancomycin andfungizone to passage three. Cell lines are tested periodically for thepresence of mycoplasma contamination using the Hoechst fluorescent stainfor mycoplasma DNA. Cells are utilized usually only until passage 10.

Cytopathic Effect Inhibition Assay:

Low passage (3-10) human foreskin fibroblast (HFF) cells aretrypsinized, counted, and seeded into 96 well tissue culture plates at acell concentration of 2.5×104 cells in 0.1 ml of MEM supplemented with10% FBS. The cells are then incubated for 24 hours at 37° C. in a 5%CO₂— 95% air, 90% humidified atmosphere. The media is then removed and100 μl of MEM containing 2% FBS is added to all but the first row. Inthe first row, 125 μl of media containing the experimental compound isadded in triplicate wells. Media alone is added to both cell and viruscontrol wells. The compound in the first row of wells is then dilutedserially 1:5 throughout the remaining wells by transferring 25 μl usinga Beckman Bio-Mek Liquid Handling Machine. The plates are then incubatedfor 60 minutes and 100 μl of an appropriate virus concentration added toeach well, excluding cell control wells which received 100 μl of MEM.For HSV-1 and HSV-2 assays, the virus concentration utilized is 1000Plaque Forming Units (PFU) per well. For CMV and VZV assays, the virusconcentration added is 2500 and 1000 PFU per well, respectively. Theplates are then incubated at 37° C. in a CO₂ incubator for three daysfor HSV-1 and HSV-2, 10 days for VZV, or 14 days for CMV. After theincubation period, the media is aspirated and the cells are stained witha 0.1% crystal violet in formalin solution for 4 hours. The stain isthen removed and the plates rinsed using tap water until all excessstain is removed. The plates are allowed to dry for 24 hours and theamount of CPE in each row determined using a BioTek MultiplateAutoreader. EC₅₀ and IC₅₀ values are determined by comparing compoundtreated and untreated cells using a computer program.

Plaque Production Assay for HSV-1 and HSV-2:

Two days prior to use, HFF cells are trypsinized, counted, and platedinto six well plates and incubated at 37° C. with 5% CO₂ and 90%humidity. On the date of assay, the compound is made up at twice thedesired concentration in 2×MEM and then serially diluted 1:5 in 2×MEM togive six concentrations of compound. The compound concentrationsutilized are usually 200 μg/ml down to 0.06 μg/ml. The virus to be usedis diluted in MEM containing 10% FBS to a desired concentration whichwill give 20-30 plaques per well. The media is then aspirated from thewells and 0.2 ml of virus is added to each well in triplicate with 0.2ml of media being added to drug toxicity wells. The plates are thenincubated for 1 hour with shaking every 15 minutes. After the incubationperiod, an equal amount of 1% agarose is added to an equal volume ofeach compound dilution. This provides final compound concentrationsbeginning with 100 μg/ml and ending with 0.03 μg/ml and a final agaroseoverlay concentration of 0.5%. The compound agarose mixture is appliedto each well in a 2 ml volume and the plates are incubated for threedays, after which the cells are stained with a 1.5% solution of neutralred. At the end of the 4-6 hours incubation period, the stain isaspirated, and plaques counted using a stereomicroscope at 10×magnification.

Plaque Production Assay for CMV:

The procedures are nearly identical to those provided for HSV with a fewminor changes. The agarose used for both the initial overlay and the twosubsequent overlays is 0.8% rather than 1%. The assay is incubated for14 days with the additional 1 ml overlays being applied on days 4 and 8.

Plaque Production Assay for VZV:

The procedures are essentially identical to those described for the HSVplaque assay with the following possible exceptions: after addition ofthe compound, the plates are incubated for ten days; on days 3 and 6, anadditional 1 ml overlay with equal amounts of 2×MEM and 1% agarose areadded.

Plaque Reduction Assay:

In certain cases, some large or highly charged molecules that are activein the CPE inhibition assay may be inactive in the plaque assay becausethe compound failed to diffuse through the agarose overlay. Therefore, amodified plaque assay can be used for confirmation, wherein the overlaymedium is liquid rather than semi-solid. The procedure for the liquidoverlay plaque assay is similar to that using the agarose overlay. Theprocedure for adding the virus is the same as for the regular plaqueassay. The compounds are made up at the desired concentrations in MEMwith 2% FBS. For HSV-1 and HSV-2 assays, an antibody preparationobtained from, e.g., Baxter Health Care Corporation is diluted 1:500 andadded to the media that the compound is diluted in to limitextracellular spread of virus through the media. For VZV and CMV, noantibody in the overlay is necessary. For the CMV and VZV assays,additional medium without the new compound is added on day five andallowed to incubate for a total of 8 and 10 days, respectively. At theend of the incubation period for all of the assays, 2 ml of a 6.0%neutral red solution is added to each well and incubated for 6 hours.The liquid is then aspirated off and plaques enumerated using astereomicroscope.

6.6.2.2 Efficacy Screening for EBV

Cells:

The two lymphoid cell lines, Raji and Daudi derived from Burkitt'slymphoma, are used. The Raji cell line is a non-producer of viral geneproducts associated with the productive viral cycle. The Daudi cell lineis a low level producer, i.e., fewer than 1% of the cells express EAspontaneously. These cells are equally susceptible to superinfection bythe P3HR-1 virus as determined by EBV VCA expression. The cells aremaintained at 37° C. in a humidified atmosphere with 5% CO₂, in culturewith RPMI-1640 medium containing 10% heat inactivated FBS, 100 u/mlPenicillin, 25 μg/ml gentamicin and 2 mM L-glutamine. The cells arepassaged twice weekly and the cell concentration adjusted to 2×10⁶/mlfor use.

Virus:

The following prototypes of infectious EBV can be used: (1) one derivedfrom supernatant fluids of the P3HR-1 cell line, which producesnon-transforming virus that induces the production of VCA after primaryinfection or superinfection of B cell lines; and (2) B95-8 virus, whichimmortalizes cord blood lymphocytes and induces tumors in marmosets, butdoes not induce an abortive productive infection even in cell linesharboring EBV genome copies. As an example, for virus production, P3HR-1cells are cultured at a concentration of 2×10⁵/ml for two weeks inmedium containing 2% FCS at 34° C. in a humidified atmosphere with 5%CO₂. Concentrated virus then is prepared from the supernatant of theculture by centrifugation at 12,000 g for 90 minutes in a SorvallCentrifuge. The pellets are resuspended in RPMI-1640 medium at 1/100 ofthe original volume and stored at −70° C.

Antibodies:

Murine monoclonal antibody to EBV VCA (Chemicon International, Inc.,Temecula, Calif.), is used in immunofluorescence assays and ELISA.Optimal monoclonal antibody concentration is determined by antibodytitration for each assay system. For single fluorochrome analysesFITC-labelled goat anti-mouse total IgG (Southern BiotechnologyAssociates, Birmingham, Ala.) is used as the second antibody.

EBV Superinfection and Compound Treatment:

Superinfection is initiated by the incubation of 0.5 ml of anappropriate concentration of EBV with 106 cells/tube in a total of 1ml/tube. In most cases, this amounts to a multiplicity of infection(MOI) of 0.1-0.2 based on VCA induction in Daudi cells. After adsorptionat 37° C. for 1 hour, 3 ml of RPMI-1640 medium is added. The cells arepelleted by centrifugation and supernatants discarded. Compoundconcentrations (0.08, 0.4, 2, 10, 50 μg/ml) in 4 ml of RPMI-1640 areadded to the appropriate tubes. RPMI-1640 is added to positive andnegative control tubes and each compound concentration is added to Daudicells without virus for toxicity controls. After incubation, the cellsin each tube are counted using a Coulter Counter and washed three timeswith phosphate buffered saline solution (PBS) (without Ca and Mg). Eachcell suspension is adjusted to a concentration of 4.0×10⁶ cells/ml inPBS. For EBV IFA and DNA hybridization assays, two sets of slides areprepared with 4×10⁴ cells/spot for each cell suspension, and air-driedovernight.

Immunifluorescence Assay:

The infected and compound treated cells are counted and washed threetimes with PBS. Cells, 4×10⁴ in PBS, are spotted on multiwell slides andair dried. The cells are then fixed for 10 minutes in acetone, washed inPBS and stained for immunofluorescence with the mouse monoclonalantibodies and FITC-labeled goat anti-mouse IgG. EBV VCA specificantibodies are used in the immunofluorescence assays. FITC-labeled goatanti-mouse IgG (Southern Biotechnology Associates, Birmingham, Ala.) isused as the second antibody. The slides are counterstained with 0.1%Evan's blue for 5 minutes and mounted with 10% glycerin in PBS. Thenumber of FITC-positive cells on each smear is determined using a Nikonfluorescence microscope. Five hundred cells are counted in each spot.The number of cells expressing EBV VCA is calculated by multiplying thefraction of antigen positive cells by the number of cells/ml in theculture at the time of harvest. The compound concentration is plottedagainst the number of antigen positive cells/ml using a computerprogram, and EC₅₀ and EC₉₀ values are calculated.

ELISA:

Daudi cells infected with P3HR-1 virus and treated with drug areharvested by centrifugation and washed three times with PBS. The cellsare pelleted and suspended to a concentration of 4×10⁶ cells/ml in PBS.One hundred μl of each suspension is dispensed in triplicate into a96-well plate, air-dried and fixed with 95% ethanol and 5% acetic acid.Uninfected cells are prepared in the same manner and used as controls.After washing the plate, primary and secondary antibodies diluted in 1%bovine serum albumin containing 0.05% Tween-20 are added sequentially toeach well and incubated at room temperature. Antibody additions areseparated by 3 washes with PBS containing 0.005% Tween-20.O-phenyldiamine (OPD) substrate is added and the reaction stopped with3N H₂SO₄ after about 10 minutes. The optical density is measured at 492nm and the EC₅₀ extrapolated using the computer software programdescribed herein.

Evaluation of Anti-Viral Agents Against EBV DNA Replication:

The Enzo Simply Sensitive Horseradish Peroxidase-AEC In Situ DetectionSystem for EBV (Enzo Diagnostics, Farmingdale, N.Y.) is used todetermine anti-viral activity against DNA synthesis. Detection andstaining are performed according to the manufacturer's instructions.Three days after superinfection and compound treatment, slides areprepared with 4×10⁴ cells/spot for each cell suspension, and air-driedovernight. The slides are fixed in acetone for 10 minutes. A biotinlabelled EBV probe is added to each spot of fixed cells and the slide iscovered with a glass coverslip. The slide is then heated on a hot plateat 95° C. for 5 minutes. After heating, the slide is placed at 37° C. ona slide warmer for 30-60 minutes for the DNAs to anneal. The coverslipsare then removed and the Post Hybridization Reagent is added to eachspot. After incubation for 10 minutes and rinsing with washing buffer,Detection Reagent is applied. The Detection Reagent is left on the slidefor 30-60 minutes on a slide warmer and then washed off with washingbuffer. Chromogen Substrate Solution is added and incubated for 20minutes on a slide warmer. The slides are washed and counter stainedwith Blue Counterstain. The slides are then rinsed with deionized waterand mounted with water. The slides are viewed in a light microscopeunder a magnification of 400×. Positive cells appear as red spots. Allthe cells are counted in several fields. The fraction of red spots inthe total number of cells counted multiplied by 100 reflects the percenthybridization.

Primary Infection Assay:

The primary infection of umbilical cord blood lymphocytes with thetransforming strain B95-8 of EBV induces the expression of thevirus-associated nuclear antigen (EBNA) in the cell. It is also knownthat B95-8 virus induces cellular DNA synthesis after infection of CBL.The availability of EBNA virus-infected cells in culture allows for theidentification and quantitation of EBV-positive cell antigens byindirect IFA staining and FACS. Cord blood lymphocytes separated byficoll-hypaque gradient are cultured in complete RPMI-1640. TheEBV-B95-8 is produced by incubating the B95-8 cell line in RPMI-1640plus 10% fetal calf serum for 10-14 days. The supernatant is collectedand stored at 0-4° C. One million CBL are infected by incubation with 1ml of the B95-8 supernant for 1 hour. The virus is removed bycentrifugation. After one wash with RPMI-1640, the infected cells aretreated with anti-viral compounds as described earlier for P3HR-1superinfection. The cell cultures are incubated for 4-6 days. Cellharvesting and immunofluorescent staining is the same as describedabove.

6.6.2.3 Efficacy Screening for HHV-6 and HHV-8

Cord Blood Lymphocytes (CBL) Cells: Fresh heparinized umbilical cordblood can be obtained, e.g., from the University of Alabama atBirmingham Hospital, and diluted 1:1 with Hank's balanced salt solutionand layered on a Histopaque 1077 (Sigma Chemical Co., St. Louis, Mo.)gradient. The tubes are centrifuged at 1600 rpm for 30 minutes at roomtemperature and serum is carefully aspirated off. The lymphocytes areremoved, washed with Hank's balanced salt solution and centrifuged at1200 rpm for 10 minutes. The supernatant is aspirated, and the cells areresuspended in RPMI 1640 containing 10% heat-inactivated FBS, 2 mML-glutamine, 100 U/ml penicillin, 0.25 μg/ml fungizone, 25 μg/mlgentamicin, 0.1 U/ml Interleukin-2 (Sigma, St. Louis, Mo.) and 0.5 μg/mlPhaseolus Vulagaris agglutinin protein (PHAP). CBLs are used in theHHV-6, Z-29 (Variant B) assays.

Human T Cell Lymphoblastoid Line HSB-2:

The HSB-2 cells can be obtained through, e.g., the NIH AIDS Research andReference Reagent Program (Rockville, Md.), and are propagated in RPMI1640 containing 10% heat-inactivated FBS, 100 U/ml penicillin, 25 μg/mlgentamicin and 2 mM L-glutamine. The cells are split 1:5 in a 175 cm²flask every 3-4 days and used in the HHV-6, GS (Variant A) assays.

Body Cavity-Based Lymphoma (BCBL-1) Cells:

BCBL-1 cells (NIH AIDS Research and Reference Program, Rockville, Md.)propagated in RPMI 1640 media containing 10% FBS, 2 mM L-Glutamine, 10μM β-Mercaptoethanol 100μ/l penicillin, and 25 μg/ml gentamicin areutilized in the HHV-8 assay.

Viruses:

There are two variants of HHV-6 known as type A variants or type Bvariants. An example of HHV-6 type A variant is the GS strain which ispropagated in the HSB-2 cells and can be obtained through, e.g., theAIDS Research and Reference Reagent Program, Division of AIDS, NIAID,NIH. These cells, referred to as HSB-2/HHV-6GS, are maintained at 5×10⁵cells/ml under the same conditions and in the same media as theuninfected HSB-2 cells. The cells are split every 3-4 days by additionof uninfected cells at 9 parts to 1 part infected cells. Stock titers ofthis virus of 1×10⁵ both in cell-associated and cell-free virus can beobtained by growth for 5 days. An example of HHV-6 type B variant is Z29(ATCC, Rockville, Md.) which is grown as a stock in CBL by incubationfor 10 days followed by collection, centrifugation and freezing of thesupernatant. HHV-8, latently expressed in the primary effusion lymphomaderived BCBL-1 cell line (NIH AIDS Research and Reference Program,Rockville, Md.) is induced into lytic HHV-8 expression by addition of100 ng/ml phorbol 12-myristate 13-acetate. BCBL-1 cells are cultured inRPMI 1640 media containing 10% FBS, 2 mM L-glutamine, 10 μMβ-mercaptoethanol 100 U/ml penicillin and 25 μg/ml gentamicin.

Primary Antibodies:

The primary antibodies used for the indirect IFA and FACS are selectedfor their antigen specificity, low cross-reactivity with other herpesviruses and fluorescence intensity as monitored by FACS. Monoclonalantibodies selected for use in the HHV-6 assay systems are screened forvariant specificity and demonstrated no A or B variant cross-reactivityin the assay systems. Monoclonal antibody 8532 (Chemicon, Temecula,Calif.) targets HHV-6 induced early nuclear proteins and is used as aprimary antibody in the HHV-6GS assay systems at a 5 μg/mlconcentration. Monoclonal antibody 8535 (Chemicon, Temecula, Calif.)which targets a B variant 101 kDa virion protein is used as a primaryantibody in the HHV-6Z-29 assay system at a 5 μg/ml concentration. TheHHV-8 monoclonal antibody KS8.1 (Bala Chandran, University of KansasDepartment of Microbiology, Molecular Genetics and Immunology) targetsthe HHV-8 viral envelope associated glycoprotein 8.1 expressed in thelate lytic phase of HHV-8 replication (Zoeteweij et al., 1999) and isused at approximately 5 μg/ml. Monoclonal antibody to the EBV VCAglycoprotein 125 (Chemicon, Temecula, Calif.) is used at a concentrationof 2.5 μg/ml for ELISA and 5 μg/ml for IFA.

Efficacy Against HHV-6:

Serial 5-fold dilutions of drug starting at 50 μg/ml are prepared inmedia. CDV is used as a positive control. Samples for determininganti-viral efficacy are prepared by incubating 1×10⁶ cells for one hourwith sufficient virus to infect approximately 35% of the cells. Afterinfection, the appropriate dilution of compound is added and cellsincubated for 4 to 6 days at 37° C. Virus free controls are prepared byincubating 1×10⁶ cells in compound-free media for the designated periodand virus controls are prepared by incubating 1×10⁶ cells for one hourwith sufficient virus to infect 35% of the cells followed by incubationin compound-free media for the designated period. After incubation, thecells are rinsed with PBS and permeabilized overnight in methanol at −80C for use in FACS.

FACS Assay:

Cells are rinsed thoroughly with PBS and a blocking solution containing5% FBS, 4% Normal goat serum (NGS) and 0.5% DMSO. Cells are thenincubated with the appropriate monoclonal antibody (HHV-6 early nuclearproteins (Chemicon, Temecula, Calif.) for HHV-6, GS variant A, a 101 kDavirion protein (Chemicon, Temecula, Calif.) for HHV-6, Z-29 variant B,and KS8.1 for HHV-8 (Bala Chandran, University of Kansas, Department ofMicrobiology, Molecular Genetics and Immunology).

6.6.2.4 Toxicity Screening for Herpes Viruses

Neutral Red Uptake Assay—HFF Cells:

Twenty-four hours prior to assay, HFF cells are plated into 96 wellplates at a concentration of 2.5×10⁴ cells per well. After 24 hours, themedia is aspirated and 125 μl of each compound concentration is added tothe first row of wells and then diluted serially 1:5 using the automatedBio-Mek Liquid Handling System in a manner similar to that used in theCPE assay. The plates are then incubated in a CO₂ incubator at 37° C.for seven days. At this time the media/compound is aspirated and 200μl/well of 0.01% neutral red in DPBS is added. This mixture is incubatedin the CO₂ incubator for 1 hour. The compound is aspirated and the cellsare washed using a Nunc Plate Washer. After removing the DPBS wash, 200μl/well of 50% ETOH/1% glacial acetic acid (in H₂O) is added. The platesare rotated for 15 minutes and the optical densities are read at 550 nmon a plate reader. CC₅₀ values are calculated using a computer program.

Cell Proliferation Assay—HFF Cells:

Twenty-four hours prior to assay, HFF cells are seeded in 6-well platesat a concentration of 2.5×10⁴ cells per well in MEM containing 10% FBS.On the day of the assay, test compounds are diluted serially in MEMcontaining 10% FBS at increments of 1:5 covering a range from 100 μg/mlto 0.03 μg/ml. For compounds that have to be solubilized in DMSO,control wells receive MEM containing 1.0% DMSO. The media from the wellsis then aspirated and 2 ml of each compound concentration is then addedto each well. The cells are then incubated in a CO₂ incubator at 37° C.for 72 hours. At the end of this time, the media-compound solution isremoved and the cells are washed. One ml of 0.25% trypsin is added toeach well and incubated until the cells start to come off of the plate.The cell-media mixture is then pipetted up and down vigorously to breakup the cell suspension, and 0.2 ml of the mixture is added to 9.8 ml ofIsoton III and counted using a Coulter Counter. Each sample is counted 3times with 2 replicate wells per sample.

MTS Tetrazolium Cytotoxicity Assay:

Serial 5-fold dilutions of test compound starting at 50 μg/ml areprepared in media and added to 1×10⁶ cells. Controls are prepared byincubating 1×10⁶ cells in compound-free media. After an incubationperiod of 3-6 days depending on the assay system, 200 μl is transferredto a 96 well plate in duplicate. 20 μl of MTS is added, and the plate iswrapped in foil and incubated at 37° C. for 4 hours. MTS is bioreducedby dehydrogenase enzymes found in metabolically active cells into anaqueous soluble formazan. The quantity of formazan product as measuredby the amount of 490 nm absorbance is directly proportional to thenumber of living cells in culture. Compound concentration is plottedagainst the optical density of each sample and CC₅₀ values werecalculated using MacSynergy II.

Cell Proliferation Assay—HSB-2 and Daudi Cells:

Serial 5-fold dilutions of compound starting at 50 μg/ml are prepared inmedia and added to 1×10⁶ cells. Controls are prepared by incubating1×10⁶ cells in compound-free media. After an incubation period of 3-4days depending on the assay system, a Coulter Counter is used todetermine the total number of cells for each sample (HSB-2 and Daudicell lines). Compound concentration is plotted against the totalconcentration of cells for each sample and IC₅₀ values are calculatedusing MacSynergy II.

Bone Marrow Assay:

In vitro toxicity can be determined by inhibition of myeloid[colony-forming units granulocyte/macrophage (CFU-GM)] and erythroid[burst-forming unit-erythroid (BFU-E)] colony formation in soft agarclonal assays. Using a 21-23 gauge needle attached to a syringe, rodentbone marrow cells are collected from the leg bone of rats or mice byflushing with Isocoves' Modified Dulbecco's medium (IMDM). A single cellsuspension is obtained by repeated aspiration through the needle.Nucleated cells are enumerated with a hemacytometer and adjusted to thedesired cell concentration in IMDM. Murine CFU-GM assays are preparedwith 2.5×10⁵ nucleated cells/ml, 20% FBS, 10 ng/ml rmGM-CSF, and 0.2%agarose. BFU-E cultures include 30% FBS, 1% deionized BSA, 0.1 mM 2-ME,4 U/ml rhEpo, 10 ng/ml rmIL-3, 2.5×105 nucleated cells/ml and 0.2%agarose (140). Triplicate wells (in 6 well plates) containing 0.1 ml ofcompound (10×) receive 1 ml of either culture mixture for eachconcentration group and slowly mixed. The cultures are allowed to gel at4° C. and then incubated for 7 (CFU-GM) or 9 (BFU-E) days at 37° C. in ahumidified atmosphere of 5% CO₂ in air. Colonies are counted using aninverted microscope. CFU-GM colonies are identified as cell clonescontaining at least 40 cells. BFU-E cultures are stained withdianisidine, and aggregates of greater than 60 hemoglobin-containingcells are counted as erythroid colonies. The median inhibitoryconcentration (IC₅₀) and the 90% inhibitory concentration (IC₉₀) arederived from linear regression analysis of the logarithm of compoundconcentration versus CFU-GM or BFU-E survival fraction.

6.6.3 In Vitro Laboratory Procedures for Assays for Influenza,Respiratory and Other Viruses

6.6.3.1 Screening Efficacy for RSV, PIV and Flu, Measles, Rhino, Adeno,SARS, VEE, Yellow Fever, West Nile, Pichinde, Punta Toro and DengueViruses

Rapid Screening Assay:

When relatively large numbers (10 or more) of test compounds areavailable, the compounds are evaluated in a 2-concentration test. Inthis procedure, two concentrations (e.g., 200 and 20 μg/ml) are tested.Compounds are diluted 1:2 when virus is added, making finalconcentrations 100 and 10 μg/ml. The standard CPE test uses an 18 hourmonolayer (80-100% confluent) of the appropriate cells, medium isdrained and each of the concentrations of test compound or placebo areadded, followed within 15 minutes by virus or virus diluent. Two wellsare used for each concentration of compound for both anti-viral andcytotoxicity testing. The plate is sealed and incubated the standardtime period required to induce near-maximal viral CPE. The plate is thenstained with neutral red by the method described below, and thepercentage of uptake indicating viable cells read on a microplateautoreader at dual wavelengths of 405 and 540 nm, with the differencetaken to eliminate background. An approximated virus-inhibitoryconcentration, 50% endpoint (EC₅₀) and cell-inhibitory concentration,50% endpoint (IC₅₀) are determined from which a general selectivityindex is calculated: SI=(IC₅₀)/(EC₅₀). An SI of 3 or greater typicallyindicates confirmatory testing is needed.

Inhibition of Cytopathic Effect (CPE):

This test, run in 96 well flat-bottomed microplates, is used for theinitial anti-viral evaluation of all new test compounds. In this CPEinhibition test, four log₁₀ dilutions of each test compound (e.g., 1000,100, 10, 1 μg/ml) are added to 3 cups containing the cell monolayer;within 5 minutes, the virus is then added and the plate sealed,incubated at 37° C. and CPE read microscopically when untreated infectedcontrols develop a 3 to 4+ CPE (approximately 72 to 120 hours). A knownpositive control compound is evaluated in parallel with test compoundsin each test. The positive control compound, for example, is: ribavirinfor dengue, influenza, measles, RSV, PIV, Pichinde, Punta Toro and VEEviruses; cidofovir for adenovirus; pirodovir for rhinovirus;6-azauridine for West Nile and yellow fever viruses; and alferon(interferon alfa-n3) for SARS virus. Follow-up testing with compoundsthat are found active in initial screening tests are run in the samemanner except 8 one-half log₁₀ dilutions of each compound are used in 4cups containing the cell monolayer per dilution. The data are expressedas 50% effective concentrations (EC₅₀).

Increase in Neutral Red (NR) Dye Uptake:

This test is run to validate the CPE inhibition seen in the initialtest, and utilizes the same 96-well micro plates after the CPE has beenread. Neutral red is added to the medium; cells not damaged by virustake up a greater amount of dye, which is read on a computerized microplate autoreader. For example, the method as described by McManus, Appl.Environment. Microbiol. 1976, 31:35-38, can be used. An EC₅₀ isdetermined from this dye uptake.

Decrease in Virus Yield:

Compounds considered active by CPE inhibition and by NR dye uptake arere-tested if additional, fresh material is available, using both CPEinhibition and, using the same plate, the effect on reduction of virusyield by assaying frozen and thawed eluates from each cup for virustiter by serial dilution onto monolayers of susceptible cells.Development of CPE in these cells is the indication of presence ofinfectious virus. As in the initial tests, a known active compound isrun in parallel as a positive control. The 90% effective concentration(EC₉₀), which is that test compound concentration that inhibits virusyield by 1 log₁₀, is determined from these data.

6.6.3.2 Screening Toxocity for RSV, PIV and Flu, Measles, Rhino, Adeno,SARS, VEE, Yellow Fever, West Nile, Pichinde, Punta Toro and DengueViruses

Visual Observation:

In the CPE inhibition tests, two wells of uninfected cells treated witheach concentration of test compound are run in parallel with theinfected, treated wells. At the time CPE is determined microscopically,the toxicity control cells are also examined microscopically for anychanges in cell appearance compared to normal control cells run in thesame plate. These changes can be enlargement, granularity, cells withragged edges, a filmy appearance, rounding, detachment from the surfaceof the well, or other changes. These changes are given a designation ofT (100% toxic), PVH (partially toxic-very heavy-80%), PH (partiallytoxic-heavy-60%), P (partially toxic-40%), Ps (partiallytoxic-slight-20%), or 0 (no toxicity-0%), conforming to the degree ofcytotoxicity seen. A 50% cell inhibitory (cytotoxic) concentration(IC₅₀) is determined by regression analysis of these data.

Neutral Red Uptake:

In the neutral red dye uptake phase of the anti-viral test describedabove, the two toxicity control wells also receive neutral red and thedegree of color intensity is determined spectrophotometrically. Aneutral red IC₅₀ (NR IC₅₀) is subsequently determined.

Viable Cell Count:

Compounds considered to have significant anti-viral activity in theinitial CPE and NR tests are re-tested for their effects on cell growth.In this test, 96-well tissue culture plates are seeded with cells(sufficient to be approximately 20% confluent in the well) and exposedto varying concentrations of the test drug while the cells are dividingrapidly. The plates are then incubated in a CO₂ incubator at 37° C. for72 hours, at which time neutral red is added and the degree of colorintensity indicating viable cell number is determinedspectrophotometrically; an IC₅₀ is determined by regression analysis.

Data Analysis: Each test compound's anti-viral activity is expressed asa selectivity index (SI), which is the IC₅₀ or IC₉₀ divided by the EC₅₀.Generally, an SI of 10 or greater is indicative of positive anti-viralactivity, although other factors, such as a low SI for the positivecontrol, are also taken into consideration. Compounds having SI valuesof 10 or greater can be evaluated against additional strains of theoriginal virus inhibited in order to more fully determine the spectrumof anti-viral activity of the compound.

6.6.4 General Procedures for Assays for Orthopoxviruses

To quickly screen out compounds that do not have activity against any ofthe herpes viruses, or are too toxic to evaluate, an assay such as aCPE-inhibition assay that is semi-automated is commonly used initiallyto screen out the negative compounds. Typically, all screening assaysare conducted in low passage human cells, and each assay system containsa positive control (CDV) and a negative control (ACV). Efficacy isdemonstrated by at least two different assay systems that detectfunctional biologic activity and should be confirmed using low passagedclinical isolates and drug resistant mutants whenever available. In thecase of Vaccinia virus (VV) and Cowpox virus (CV), efficacy against VVand CV is confirmed using other isolates. Toxicity is determined usingboth resting and proliferating human fibroblast cells and proliferatinglymphoblastic cells, and for selected compounds, toxicity in rodentmyeloid and erythroid progenitor cells is assessed.

6.6.4.1 Screening Assays for VV and CV

Compounds are initially screened for activity using the CPE assay in HFFcells. Further testing in two other cells lines, Vero and MRC-5, andagainst other strains of virus is possible for compounds thatdemonstrate activity in other assay systems. The screening assay systemsutilized are selected to show specific inhibition of a biologicfunction, i.e., cytopathic effect (CPE) in susceptible human cells. Inthe CPE-inhibition assay, test compound is added 1 hour prior toinfection so the assay system will have maximum sensitivity and detectinhibitors of early replicative steps such as adsorption or penetrationas well as later events. To rule out non-specific inhibition of virusbinding to cells, all compounds that show reasonable activity in the CPEassay are confirmed using a classical plaque reduction assay in whichthe drug is added 1 hour after infection. These assay systems also canbe manipulated by increasing the pretreatment time in order todemonstrate anti-viral activity with oligodeoxynucleotides and/orpeptides. By delaying the time of addition of compound after infection,information regarding which step in the virus life cycle is inhibited(i.e., early vs. late functions) can be gained. A direct inactivationassay can be employed to determine the virucidal activity of selectedcompounds.

Efficacy:

In the assays used for primary screening, a minimum of six compoundconcentrations is typically used, covering a range of, e.g., 100 mg/mlto 0.03 mg/ml, in 5-fold increments. These data allow for creating doseresponse curves. From these data, the dose that inhibited viralreplication by 50% (effective concentration 50; EC₅₀) is usuallycalculated using a computer software program, for example, MacSynergy IIby M. N. Prichard, K. R. Asaltine, and C. Shipman, Jr., University ofMichigan, Ann Arbor, Mich.

Toxicity:

The same compound concentrations used to determine efficacy are alsoused on uninfected cells in each assay to determine toxicity of eachexperimental compound. The compound concentration that is cytotoxic tocells as determined by their failure to take up a vital stain, neutralred (cytotoxic concentration 50; CC₅₀), is determined as describedabove. A neutral red uptake assay can be used. The assay is reproducibleand allows quantitation of toxicity based on the number of viable cellsrather than cellular metabolic activity. In some cases, the toxicity ofnew compounds on dividing cells is determined at a very early stage oftesting. A cell proliferation assay using HFF cells is a sensitive assayfor detecting compound toxicity to dividing cells. The compoundconcentration that inhibits cell growth by 50% (IC₅₀) is calculated asdescribed above. In comparison with four human diploid cell lines andVero cells, HFF cells are known to be very sensitive and predictive oftoxicity for bone marrow cells.

6.6.4.2 Confirmation Assays for VV and CV

Anti-Viral Activity:

Compounds that show activity in the CPE-inhibition assay are confirmedusing the plaque reduction assay. Susceptibility of additional virusstrains of VV, CV and activity in other cell types can also bedetermined for selected compounds.

Toxicity:

In addition to the toxicity component incorporated into each assaysystem, a standardized cell cytotoxicity assay using a vital stainuptake (Neutral Red) is performed using 7 days of compound exposure toconfluent non-dividing cells. This assay measures direct cellcytotoxicity (CC₅₀). In this regard, a neutral red uptake assay isreproducible and allows for quantitation of toxicity based on the numberof viable cells rather than cellular metabolic activity. In some cases,the toxicity of new compounds on dividing cells is determined at a veryearly stage of testing. A cell proliferation assay using HFF cells is asensitive assay for detecting compound toxicity to dividing cells, andthe compound concentration that inhibits cell growth by 50% (IC₅₀) iscalculated as described above.

6.6.5 In Vitro Laboratory Procedures for Assays for Orthopoxviruses

6.6.5.1 Efficacy Screening for VV and CV

Preparation of Human Foreskin Fibroblast (HFF) Cells:

Newborn human foreskins are obtained as soon as possible aftercircumcision and placed in minimal essential medium (MEM) containingvancomycin, fungizone, penicillin, and gentamicin at the usualconcentrations, for 4 hours. The medium is then removed, the foreskinminced into small pieces and washed repeatedly with phosphate bufferedsaline (PBS) deficient in calcium and magnesium (PD) until red cells areno longer present. The tissue is then trypsinized using trypsin at 0.25%with continuous stirring for 15 minutes at 37° C. in a CO₂ incubator. Atthe end of each 15-minute period, the tissue is allowed to settle to thebottom of the flask. The supernatant containing cells is poured throughsterile cheesecloth into a flask containing MEM and 10% fetal bovineserum. The flask containing the medium is kept on ice throughout thetrypsinizing procedure. After each addition of cells, the cheesecloth iswashed with a small amount of MEM containing serum. Fresh trypsin isadded each time to the foreskin pieces and the procedure repeated untilall the tissue is digested. The cell-containing medium is thencentrifuged at 1000 RPM at 4° C. for 10 minutes. The supernatant liquidis discarded and the cells are resuspended in a small amount of MEM with10% FBS. The cells are then placed in an appropriate number of 25 cm²tissue culture flasks. As cells become confluent and needtrypsinization, they are expanded into larger flasks. The cells are kepton vancomycin and fungizone to passage four, and maintained onpenicillin and gentamicin. Typically, cells are used only throughpassage 10.

Cytopathic Effect Inhibition Assay:

Low passage HFF cells are seeded into 96 well tissue culture plates 24hours prior to use at a cell concentration of 2.5×10⁵ cells per ml in0.1 ml of MEM supplemented with 10% FBS. The cells are then incubatedfor 24 hours at 37° C. in a CO₂ incubator. After incubation, the mediumis removed and 125 ml of experimental compound is added to the first rowin triplicate wells, all other wells having 100 ml of MEM containing 2%FBS. The compound in the first row of wells is then diluted serially 1:5throughout the remaining wells by transferring 25 ml using the BioMek2000 Laboratory Automation Workstation. After dilution of the compound,100 ml of the appropriate virus concentration is added to each well,excluding cell control wells, which received 100 ml of MEM. The virusconcentration utilized is 1000 PFU's per well. The plates are thenincubated at 37° C. in a CO₂ incubator for 7 days. After the incubationperiod, media is aspirated and the cells stained with a 0.1% crystalviolet in 3% formalin solution for 4 hours. The stain is removed and theplates rinsed using tap water until all excess stain is removed. Theplates are allowed to dry for 24 hours and then read on a BioTekMultiplate Autoreader at 620 nm. The EC₅₀ values are determined bycomparing compound treated and untreated cells using a computer program.

Plaque Reduction Assay:

Two days prior to use, HFF cells are plated into 6 well plates andincubated at 37° C. with 5% CO₂ and 90% humidity. On the date of assay,the compound is made up at twice the desired concentration in 2×MEM andthen serially diluted 1:5 in 2×MEM using 6 concentrations of compound.The initial starting concentration is usually 200 mg/ml down to 0.06mg/ml. The virus to be used is diluted in MEM containing 10% FBS to adesired concentration which will give 20-30 plaques per well. The mediais then aspirated from the wells, and 0.2 ml of virus is added to eachwell in duplicate with 0.2 ml of media being added to drug toxicitywells. The plates are then incubated for 1 hour with shaking every 15minutes. After the incubation period, an equal amount of 1% agarose isadded to an equal volume of each compound dilution. This addition givesfinal compound concentrations beginning with 100 mg/ml and ending with0.03 mg/ml and a final agarose overlay concentration of 0.5%. Thecompound/agarose mixture is applied to each well in 2 ml volume and theplates are incubated for 3 days, after which the cells are stained witha 0.01% solution of neutral red in phosphate buffered saline. After a5-6 hours incubation period, the stain is aspirated, and plaques countedusing a stereomicroscope at 10× magnification.

6.6.5.2 Toxicity Screening for VV and CV

Neutral Red Uptake Assay:

Twenty-four hours prior to assay, HFF cells are plated into 96 wellplates at a concentration of 2.5×10⁴ cells per well. After 24 hours, themedia is aspirated and 125 ml of compound is added to the first row ofwells and then diluted serially 1:5 using the BioMek 2000 LaboratoryAutomation Workstation in a manner similar to that used in the CPEassay. After compound addition, the plates are incubated for 7 days in aCO₂ incubator at 37° C. At this time, the media/compound mixture isaspirated and 200 ul/well of 0.01% neutral red in PBS is added. Thismixture is incubated in the CO₂ incubator for 1 hour. The dye isaspirated and the cells are washed using a Nunc Plate Washer. Afterremoving the PBS, 200 mg/well of 50% ETOH/1% glacial acetic acid (inH₂O) is added. The plates are rotated for 15 minutes and the opticaldensities read at 540 nm on a plate reader. The EC₅₀ values aredetermined by comparing compound treated and untreated cells using acomputer program.

Cell Proliferation Assay:

Twenty-four hours prior to assay, HFF cells are seeded in 6-well platesat a concentration of 2.5×10⁴ cells per well in MEM containing 10% FBS.On the day of the assay, compounds are diluted serially in MEMcontaining 10% FBS at increments of 1:5 covering a range from 100 mg/mlto 0.03 mg/ml. For drugs that have to be solubilized in DMSO, controlwells receive MEM containing 1% DMSO. The media from the wells isaspirated, and 2 ml of each drug concentration is then added to eachwell. The cells are incubated in a CO₂ incubator at 37° C. for 72 hours.At the end of this time, the media-compound solution is removed and thecells washed. One ml of 0.25% trypsin is added to each well andincubated until the cells start to come off of the plate. The cell-mediamixture is then pipetted up and down vigorously to break up the cellsuspension and 0.2 ml of the mixture is added to 9.8 ml of Isoton IIIand counted using a Coulter Counter. Each sample is counted 3 times with2 replicate wells per sample.

Bone Marrow Clonogenic Assay:

In vitro toxicity to bone marrow progenitor cells can be determined byinhibition of myeloid [colony-forming units granulocyte/macrophage(CFU-GM)] and erythroid [burst-forming unit-erythroid (BFU-E)] colonyformation in soft agar clonal assays. Using a 21-23 gauge needleattached to a syringe, rodent bone marrow cells are collected from theleg bone of rats or mice by flushing with Isocoves' Modified Dulbecco'smedium (IMDM). A single cell suspension is obtained by repeatedaspiration through the needle. Nucleated cells are enumerated with ahemacytometer and adjusted to the desired cell concentration in IMDM.Murine CFU-GM assays are prepared with 2.5×10⁵ nucleated cells/ml, 20%FBS, 10 ng/ml rmGM-CSF, and 0.2% agarose. BFU-E cultures include 30%FBS, 1% deionized BSA, 0.1 mM 2-ME, 4 U/ml rhEpo, 10 ng/ml rmIL-3,2.5×10⁵ nucleated cells/ml and 0.2% agarose. Triplicate wells (in 6 wellplates) containing 0.1 ml of compound (10×) receive 1 ml of eitherculture mixture for each concentration group and slowly mixed. Thecultures are allowed to gel at 4° C. and then incubated for 7 (CFU-GM)or 9 (BFU-E) days at 37° C. in a humidified atmosphere of 5% CO₂ in air.Colonies are counted using an inverted microscope. CFU-GM colonies areidentified as cell clones containing at least 40 cells. BFU-E culturesare stained with dianisidine, and aggregates of greater than 60hemoglobin-containing cells are counted as erythroid colonies. Themedian inhibitory concentration (IC₅₀) and the 90% inhibitoryconcentration (IC₉₀) are derived from linear regression analysis of thelogarithm of compound concentration versus CFU-GM or BFU-E survivalfraction.

6.6.6 Assays for Hepatitis Viruses

6.6.6.1 Hepatitis B Virus (HBV)

A variety of cell-culture based anti-HBV analyses are available.Candidate compounds are initially assayed in a primary screening assay.Compounds demonstrating reasonable anti-viral and cytotoxicity profilesare then candidates for several additional follow-up analyses. For theprimary screening assay, routinely 2-3 mg are required for compoundswith molecular weights in the range of standard nucleosides (e.g.,300-500). Additional compound may be required for follow-up analyses.Molecular weights and solubility information are provided if available.If no preferred solvent is specified, 100% tissue culture DMSO will beused. Compounds are typically solubilized in aqueous solutions (normalpH range) at a minimum of a 10×final testing concentration or in DMSO ata minimum 50× test concentration. EtOH is generally not well toleratedby the cell lines used for these studies, but final concentrations ofEtOH of less than 0.03% are usually acceptable. Compounds which need tobe tested in other solvents should be accompanied by a small amount ofsolvent (under a separate accession number) to control for cytotoxicity.For compounds in solution, approximately 0.25 ml of a 100× stock isminimally required.

Primary Assay:

HBV anti-viral assays (Korba & Gerin, Antivir. Res. 1992, 19:55) areconducted using confluent cultures of 2.2.15 cells maintained on 96-wellflat-bottomed tissue culture plates (confluence in this culture systemis required for active, high levels of HBV replication equivalent tothat observed in chronically-infected individuals (Sells et al., J.Virol. 1988, 62:2836; Korba and Gerin, Antivir. Res. 1992, 19:55).HepG2-2.2.15 is a stable cell line containing the hepatitis B virus(HBV) ayw strain genome. Anti-viral compounds blocking any late step ofviral replication such as transcription, translation, pregenomeencapsidation, reverse transcription, particle assembly and release canbe identified and characterized using this cell line.

Cultures are treated with nine consecutive daily aliquots of the testcompounds. Typically, 4 doses (10-fold or 3-fold steps), in triplicateare used. HBV DNA levels in the culture medium (representing HBV virionproduction) are assessed by quantitative blot hybridization 24 hoursafter the last treatment. Alternatively, whether a compound reduces theproduction of secreted HBV from cells can be initially assessedutilizing real time quantitative PCR (TaqMan) assay to directly andaccurately measure HBV DNA copies. Cytotoxicity is assessed by uptake ofneutral red dye 24 hours following the last treatment. Lamivudine (LMV)is used as the standard assay control, but other control compounds arealso available.

EC₅₀, EC₉₀ and CC₅₀ values are calculated by linear regression analysis(MS EXCEL®, QuattroPro®) using data combined from all treated cultures(Korba & Gerin, Antivir. Res. 1992, 19:55; Okuse et al., Antivir. Res.2005, 65:23). Standard deviations for EC₅₀ and EC₉₀ values arecalculated from the standard errors generated by the regressionanalyses. EC₅₀ and EC₉₀ are compound concentrations at which a 2-fold,or a 10-fold depression of HBV DNA (relative to the average levels inuntreated cultures), respectively, is observed. CC₅₀ is the compoundconcentration at which a 2-fold lower level of neutral red dye uptake(relative to the average levels in untreated cultures) is observed. TheSelectivity index (S.I.) is calculated as CC₅₀/EC₉₀ since at least a3-fold depression of HBV DNA levels is typically required to achievestatistical significance in this assay system (Korba & Gerin, Antivir.Res. 1992, 19:55).

Secondary Assay:

Confluent cultures of 2.2.15 cells maintained on 48-well flat-bottomedtissue culture plates are treated as described for the primary assay.HBV virion DNA levels in the culture medium and cytotoxicity areassessed as described for the primary assay. In addition, intracellularHBV DNA replication is measured by quantitative Southern blothybridization analysis (Korba & Gerin, Antivir. Res. 1992, 19:55). EC₅₀,EC₉₀ and S.I. values are calculated for both virion DNA andintracellular HBV DNA replication intermediates. In certain cases,additional assays (tertiary assays) can be conducted.

Combination Studies:

Compounds are mixed at approximately equipotent concentrations and thismolar ratio is maintained during serial dilution (Korba, Antivir. Res.1996, 29:49; Iyer et al. 2004). To compensate for potential unforeseeninteractions (e.g., changes in uptake, metabolism, etc.), theconcentration of one compound is altered approximately 3-fold higher oflower relative to the second compound so that three different ratios areused in one experiment. Cultures are treated with 6-8 serial dilutionsof the mixtures, as with the corresponding monotherapies, as describedfor the primary assay. Evaluation of compound interactions in thecombination treatments is conducted against the correspondingmonotherapies in the same experiments using the Combostat® (Biosoft,Inc.) analysis software. For combination treatments, EC₅₀, EC₉₀, CC₅₀and S.I. (CC₅₀/EC₉₀) are presented for the first compound listed. Themolar ratio of the compounds in each combination is also indicated.

Alternatively, after the anti-viral activity of test compounds againstHBV is confirmed, the interactions of the compounds with 3TC, IFNα andother compounds in terms of efficacy (synergy, additivity, antagonism)and toxicity (combination toxicity) can be evaluated with the 2.2.15cells using the quantitative HBV TaqMan PCR assay.

Drug Resistant HBV:

Activity against recombinant HBV carrying clinically relevant mutationsthat confer resistance to licensed drugs is performed using transienttransfection of HBV DNA (Tatti et al., Antivi. Res. 2002, 55:27; Iyer etal., AAC 2004, 48:2199). Cultures are transfected in 48-well cultureplates with Lipofectamine 2000™ (Gibco, Inc) following themanufacturer's procedure. Beginning three days post-transfection,cultures are treated for 5 days with anti-viral compounds. Anti-viralactivity is determined by quantitative Southern Blot hybridization ofintracellular HBV DNA replication intermediates. Currently, thefollowing mutants are available: lamviudine (LMV)-resistant, polM204V,polM2041, polL180M, polM204V/L180M (Allen et al., Hepatology 1998,27:1670); adefovir dipovoxil (ADV)-resistant, polN236T (Angus et al.,Gastroenterology 2003, 125:292). Standardized nomenclature is used forHBV polymerase assignment (Stuyver et al., Hepatology 2001, 33:751).Additional mutants (TNFR, ETVR) are under construction.

Other tests can be conducted in order to evaluate the ability ofcompounds to inhibit the known 3TC- and penciclovir-resistant mutants ofHBV. Stable cell lines with control wild-type HBV and the followingmutations known to be associated with resistance of HBV to these agentscan be used: (1) L526M (rtL180M) of Domain B & YMDD M550V (rtM204V) ofDomain C (the most common mutation pattern observed during HBVbreakthrough viremia); (2) L526M alone (the most common mutationassociated with penciclovir resistance; also associated with someresistance against 3TC); and control wild-type HBV.

HBV Protein Production and RNA Transcription:

Semi-quantitative ELISA-based analysis of HBV proteins is performed(Korba & Gerin, Antivir. Res. 1995, 28:225; Korba et al., Antivir. Res.2008, 77:56) using samples diluted (2 to 10-fold) to bring levels intothe dynamic response ranges of the assays. Qualitative analysis of HBVproteins is also performed using standard Western blot techniques(Muller et al., J. Infect. Dis. 1992, 165:929). HBV surface (HBsAg) andHBV e (HBeAg) antigens are analyzed from culture medium samples, and HBVcore (HBcAg) is analyzed from intracellular lysates (normalized fortotal cell protein content in each culture sample). Intracellular HBVRNA (normalized to the level of cellular B-actin RNA in each culturesample) is assessed by quantitative northern blot hybridization (Korba &Gerin, Antivir. Res. 1995, 28:225).

HBV Mechanism of Action Studies:

A variety of assays can be used to pinpoint the mechanism of action ofanti-viral compounds. Examples include the following:

-   -   Extracellular HBV virions: In addition to the quantitative PCR        analysis, a Southern blot of the HBV particles secreted from        compound-treated cells can be performed;    -   Intracellular HBV particles: HBV particles can be isolated from        the treated 2.2.15 cells and the pregenomic RNA examined by        Southern blot analysis. This can be helpful in identifying the        site of action of a late-acting compound;    -   Intracellular HBV replicative intermediates: Nucleic acids        isolated from the cells can be examined by Southern blots to        assess the distribution of circular partially double-stranded        HBV DNA, linear partially double-stranded DNA and single        stranded HBV DNA;    -   HBV transcription: Effects on HBV genomic and subgenomic viral        RNA synthesis are studied by Northern blot and primer extension        analysis;    -   HBsAg and HBeAg release assay: ELISAs are used to quantify the        amounts of the HBV envelope protein, surface antigen (HBsAg),        and of secreted e-antigen (HBeAg) released from cultures;    -   Western blot analysis: Western blots are conducted to study HBV        core and envelope protein expression;    -   Novel mechanism of action studies: Specific effects on HBV        transcription and replication may arise from alterations in        DNA-protein interactions, sometimes affected by cellular growth        factors, at the HBV enhancers, promoters or through the        transcriptional transactivator X-protein; and    -   Endogenous Polymerase Assay: Extracellular HBV virions contain        partially double-stranded circular DNA genomes. Purified virions        are used to assay the ability of anti-viral drugs to inhibit the        endogenous polymerase activity of HBV. Normally, this activity        functions to complete (+) strand synthesis following the        infection of new cells by HBV virions.

6.6.6.2 Hepatitis C Virus (HCV) Protocol I

Cell line Huh7 ET (luc-ubi-neo/ET), which contains a new HCV RNAreplicon with a stable luciferase (LUC) reporter, is used. It is similarto the cell line 5-2 (Krieger et al., J. Virol. 2001, 75:4614-4624.),but contains additional modifications that make the cell line morerobust and provide stable LUC expression for anti-viral screening. Thecomposition of the replicon is shown diagrammatically below:

The HCV RNA replicon ET contains the 5′ NTR (IRES) of HCV 5′ whichdrives the production of a firefly luciferase (Luc), ubiquitin (Ubiq),and neomycin phosphotransferase (Neo) fusion protein. Ubiquitin cleavagereleases the LUC and Neo genes. The EMCV IRES element (E-I) controls thetranslation of the HCV structural proteins NS3-NS5. The NS3 proteincleaves the HCV polyprotein to release the mature NS3, NS4A, NS4B, NS5Aand NS5B proteins that are required for HCV replication. At the 3′ endof the replicon is the authentic 3′ NTR of HCV.

The LUC reporter is used as an indirect measure of HCV replication. Theactivity of the LUC reporter is directly proportional to HCV RNA levelsand positive control anti-viral compounds behave comparably using eitherLUC or RNA endpoints. The use of the LUC endpoint is more economicalthan HCV RNA and can be used for high-throughput applications to screenlibraries of compounds.

Primary HCV RNA Replicon Assay:

First, the effect of compounds added in triplicate at a single high-testconcentration of 20 mM on HCV RNA-derived LUC activity and cytotoxicityis examined. Human interferon alpha-2b is included in each run as apositive control compound. Subconfluent cultures of the ET line areplated out into 96-well plates that are dedicated for the analysis ofcell numbers (cytotoxicity) or anti-viral activity, and test compoundsare added to the appropriate wells the next day. Cells are processed 72hours later when the cells are still subconfluent. Compounds thatreduced the LUC signal by 50% or more relative to the untreated cellcontrols move forward to the next screening steps. A compound'scytotoxicity is assessed as the percent viable cells relative to theuntreated cell controls.

HCV RNA Replication Confirmation Assay:

The HCV RNA replicon confirmatory assay is then used to examine theeffects of compounds at, for example, five half-log concentrations each.Human interferon alpha-2b is included in each run as a positive controlcompound. Subconfluent cultures of the ET line are plated out into96-well plates that are dedicated for the analysis of cell numbers(cytotoxicity) or anti-viral activity and the next day test compoundsare added to the appropriate wells. Cells are processed 72 hours laterwhen the cells are still subconfluent. Compound EC₅₀ and EC₉₀ values(anti-viral activity) are derived from HCV RNA levels assessed as eitherHCV RNA replicon-derived LUC activity or as HCV RNA using TaqMan RT-PCR.Compound IC₅₀ and IC₉₀ values (cytotoxicity) are calculated usingCytoTox-1 (Promega), a colorimetric assay used as an indicator of cellnumbers and cytotoxicity when the LUC assay system is employed, whileribosomal (rRNA) levels determined via TaqMan RT-PCR are used as anindication of cell numbers in the RNA-based assay. Compound selectivityindices SI₅₀ and SI₉₀ values are also calculated.

6.6.6.3 Hepatitis C Virus (HCV) Protocol II

A variety of cell-culture based anti-HCV analyses are available.Candidate compounds are initially assayed in a primary screening assay.Compounds demonstrating reasonable anti-viral and cytotoxicity profilesare then candidates for several additional follow-up analyses. For theprimary screening assay, routinely 2-3 mg are required for compoundswith molecular weights in the range of standard nucleosides (e.g.,300-500). Additional compound may be required for follow-up analyses.Molecular weights and solubility information are provided if available.If no preferred solvent is specified, 100% tissue culture DMSO is used.Compounds are solubilized in aqueous solutions (normal pH range) at aminimum of a 10× final testing concentration or in DMSO at a minimum 50×test concentration. EtOH is generally not well tolerated by the celllines used for these studies, but final concentrations of EtOH of lessthan 0.03% are acceptable. Compounds which need to be tested in othersolvents should be accompanied by a small amount of solvent (under aseparate accession number) to control for cytotoxicity. For compounds insolution, approximately 0.25 ml of a 100× stock is minimally required.

Primary Assay:

Anti-viral activity against HCV is assessed in a 3-day assay (Okuse etal., Antivir. Res. 2005, 65:23; Korba et al., Antivir. Res. 2008, 77:56)using the stably-expressing HCV replicon cell line, AVA5 (sub-genomic(CON1), genotype 1b) (Blight et al., Science 2000, 290:1972) maintainedas sub-confluent cultures on 96-well plates. Anti-viral activity isdetermined by blot hybridization analysis of intracellular HCV RNA(normalized to the level of cellular B-actin RNA in each culturesample). Cytotoxicity is assessed by neutral red dye uptake in culturesmaintained in parallel plates.

EC₅₀, EC₉₀ and CC₅₀ values are calculated by linear regression analysis(MS EXCEL®, QuattroPro®) using data combined from all treated cultures(Korba & Gerin, Antivir. Res. 1992, 19:55; Okuse et al., Antivir. Res.2005, 65:23). Standard deviations for EC₅₀ and EC₉₀ values arecalculated from the standard errors generated by the regressionanalyses. EC₅₀ and EC₉₀ are compound concentrations at which a 2-fold,or a 10-fold depression of intracellular HCV RNA (relative to theaverage levels in untreated cultures), respectively, is observed. CC₅₀is the compound concentration at which a 2-fold lower level of neutralred dye uptake (relative to the average levels in untreated cultures) isobserved. The Selectivity index (S.I.) is calculated as CC₅₀/EC₉₀.Recombinant human interferon 2b (PBL laboratories, Inc.) is used as anassay control. Compounds currently in clinical trials that are directedagainst NS3 and NS5B can also be used.

Secondary Assay:

This assay assesses activity against additional genotypes using theformat described for the primary assay. Activity against the genotype 1bHCV is included for comparison. One exemplary replicon cell linecontains H/FL-Neo (genotype 1a (H77), full length construct) (Blight etal., J. Virol. 2003, 77:3181). A genotype 2a construct (J6/JFH-1, fulllength) can be used to assess for future inclusion. EC₅₀, EC₉₀, CC₅₀ andS.I. values are calculated for each replicon cell line. In someinstances, additional assays (tertiary assays) may also be conducted.

Combination Studies:

Compounds are mixed at approximately equipotent concentrations and thismolar ratio is maintained during serial dilution (Korba, Antivir. Res.1996, 29:49; Iyer et al., 2004). Usually, three different ratios areused in one experiment. Cultures are treated with 6-8 serial dilutionsof the mixtures, as with the corresponding monotherapies, as describedfor the primary assay. Evaluation of compound interactions in thecombination treatments is conducted against the correspondingmonotherapies in the same experiments using the Combostat® (Biosoft,Inc.) analysis software. For combination treatments, EC₅₀, EC₉₀, CC₅₀and S.I. (CC₅₀/EC₉₀) are presented for the first compound listed. Themolar ratio of the compounds in each combination is also indicated.

Drug-Resistant HCV:

Since no licensed anti-HCV drugs for which resistance mutations have yetbeen identified, a panel of mutants conferring resistance to compoundsin mid to late phase clinical trials is compiled. Some availablestablereplicon-containing cell lines (Korba et al., Antivir. Res. 2008, 77:56)are genotype 1 B NS5B S282T (Perra et al., Nucleosides NucleotidesNucleic Acids 2005, 24:767), and NS3 A156S and NS3 A156V (Courcambeck etal., Antivir. Ther. 2006, 11:847) drug-resistant mutants. The geneticbackground is the same as that in the BB7 replicon (AVA5 cells) used inthe primary assay. Activity against these mutants is assessed asdescribed in the primary assay, except that semi-quantitative real-timePCR is used for the analysis of HCV RNA due to reduced replicationlevels.

The genotype 1b mutants can also be assessed in this manner. For thisassay, Huh7.5 cells are transfected with HCV RNA using Liofectamine2000™ (Gibco, Inc.) in 6-well culture plates. Three dayspost-transfection, cultures are exposed to 125 g/mL G418 and testcompounds. After 10-14 days, surviving colonies are fixed, stained, andcounted. EC₅₀ and EC₉₀ values are calculated for each transfected RNA.

6.6.7 Assays for Papilloma Viruses

Assays for Human Papilloma Virus (HPV) 11 and 40:

A431 cells are plated 2×10⁵ cells/well in 6-well cluster dishes.Replicate aliquots of HPV-11 (or HPV-40) are added to each wellrepresenting an MOI of 150 particles per cell. Dilutions of compound areadded to triplicate cultures. Control wells without virus are includedand receive media alone. Positive control compound can be, e.g., HPMPC(cidofovir) at 300 μg/ml. Cell cultures are harvested, lysed with Trizolreagent (GIBCO/BRL) and RNA prepared. QRT-PCR is conducted to quantitatethe proportion of viral E1-E4 transcripts and a cellular reference RNAfor the TATA-binding protein (TBP). Anti-viral effects of compounds areassessed as an EC₅₀ value representing a 50% reduction in the amount ofE1-E4 viral transcript when compared with cultures infected with HPV-11(or HPV-40) alone. CC₅₀ toxicity is calculated as the compound dose atwhich 50% of total cellular RNA is recovered. From these two values, theSelectivity Index (SI) is determined from CC₅₀/EC₅₀. Usually, SI>5 wouldbe significant for the detection of an anti-viral activity.

The assay procedure can be modified to test compounds with microbicidalactivity if necessary. This modification is represented by drug additionto A431 cells at the same time as infectious virus.

Assays for Bovine Papilloma Virus (BPV) 1:

C127 cells are plated 3×10³ cells/well into wells of a 96-wellflat-bottomed microculture plate. Replicate aliquots of BPV-1 are addedto each well, representing approximately 100 focus-forming units.Control wells without virus are included. Dilutions of drug are added totriplicate cultures of both BPV-1-infected and uninfected cultures.Control wells receive media without compound. Positive control compoundcan be, e.g., cidofovir at 5 μg/ml. Cell cultures are fed with mediumand compound every 3-4 days. Cell numbers and viability are assessedusing the MTS assay. Anti-viral effects of compounds are calculatedusing the following formula to obtain % anti-viral activity:

B&A/B&C×100%=% Anti-viral activity

-   -   A=O.D. of BPV-1-containing, compound-treated cultures    -   B=O.D. of BPV-1 containing cell cultures    -   C=O.D. of cultures of cells alone.        The EC₅₀ value represents a 50% reduction in the amount of O.D.        values (MTS signal) of compound-treated virus-infected cultures        when compared with cultures containing BPV-1 alone and cultures        containing cells alone. The Selectivity Index (SI) is determined        from CC₅₀/EC₅₀. Usually, SI>5 would be significant for the        detection of an anti-viral activity.

The assay procedure can be modified to test compounds with microbicidalactivity if necessary. This modification is represented by drug additionto C127 cells at the same time as infectious virus.

Assays for Human Papilloma Virus (HPV) 31b:

Cultures of CIN612, clone 9E cells are prepared from known protocols.Individual rafts are developed containing multi-layers of 9E cellsgrowing on a collagen matrix impregnated with mitomycin C-treatedfibroblast cells. Rafts are treated with compound delivered into thecell culture media that can diffuse into the 9E multilayer. Treatmentsare continuous for the culture duration, which is typically a period of10 days. After 10 days culture, the 9E rafts are harvested and assayedfor HPV-31b DNA (measure of viral DNA replication) and E1-E4 viraltranscription (viral function) using the QRT-PCR assay described for theHPV-11 monolayer assay system. Primers are prepared to quantitateHPV-31B DNA and RNA (E1-E4) and the quantitation compared to TBP.Anti-viral activity is measured quantitatively as either or both areduction in viral DNA and RNA when compared to placebo-treated 9Erafts. A portion of each raft is removed for histology (H&E,immunostaining for specific marker keratins [keratin 10, involucrin]).Viral DNA and RNA levels are plotted against compound concentrations todetermine EC₅₀ (50% reduction in viral DNA and/or RNA), CC₅₀ (50%reduction in yield of total RNA/DNA). The Selectivity Index (SI) isdetermined from CC₅₀/EC₅₀. Usually, SI>5 would be significant for thedetection of an anti-viral activity.

6.6.8 Assays for BK Virus (BKV)

Assays for BK virus (BKV) can be conducted by following the proceduresdescribed in, e.g., Farasati et al., Transplantation, 2005, 79(1):116-118. Generally, the principle of the assay is to measure the effectof test compounds on the rate of viral replication by quantitativereal-time PCR for BKV viral capsid protein 1 DNA. Simultaneousquantification of a housekeeping gene such as aspartoacylase (ACY) DNApermits monitoring of host-cell replication. Regression analysis ofdose-response curve allows for determination of EC₅₀, which is definedas the compound concentration that reduces the yield of BKV DNA by 50%.The ratio of IC₅₀ to EC₅₀ (selectivity index) is used to compare theanti-viral effect of different test compounds in relation to theirsafety.

For example, anti-viral testing can be performed using BKV, Gardnerstrain (available from ATCC). The cells are expanded, e.g., in humanembryonic lung fibroblasts (WI-38 cells), using DMEM medium supplementedby 10% fetal bovine serum and L-glutamine, incubated at 37° C. under 5%CO₂. Each test compound is typically tested at least three times using abroad range of concentrations covering 4-5 orders of magnitude.Experiments usually include a negative control consisting of cellsexposed only to the diluents.

Each compound sensitivity experiment requires inoculation of 50,000log-phase WI-38 cells in six-well culture plates. After plating thecells, viral infection is achieved by the addition of 2×10³ to 2×10⁶ BKVparticles to each culture well in a volume of 0.5 mL. After a 2 hours of37° C. incubation, unbound virus is washed off with tissue culturemedium. The cultures are maintained in DMEM medium, supplemented by 10%fetal bovine serum and L-Glutamine, at 37° C., under 5% CO₂, for 7 days.Cells are harvested by 0.25% trypsin-1 mM Na-EDTA digestion at 37° C.for 10 minutes and viability assessed by Trypan blue dye exclusion test.DNA extraction on the cell lysates is performed with a commerciallyavailable kit (QIAamp DNA Mini kit, Qiagen, Valencia, Calif.). BKV VP-1DNA is amplified from the total DNA by a TaqMan quantitative PCRreaction performed in an ABI Prism 7700 Sequence Detector (ABI, FosterCity, Calif.). To keep track of variable input of cellular DNA indifferent cell-culture experiments, each cell lysate is subjected tosimultaneous TaqMan PCR for ACY.

6.6.9 Assays for Dengue Virus (DENV)

In vitro assays for DENV can be conducted using procedures substantiallysimilar to those described, for example, in Heaton et al., Proc. Natl.Acad. Sci., 2010, 107(40): 17345-17350. Huh-7.5 cells (a subline derivedfrom the hepatocyte Huh7 cell line) are maintained in DMEM-high glucosesupplemented with 0.1 mM nonessential amino acids, 5% v/v FBS, andpenicillin-streptomycin. In certain cases, DENV-luciferase replicon RNAsare introduced in Huh-7.5 cells by electroporation. At 24 hourpost-electroporation, the cells are treated with varying concentrationsof test compounds, maintained for another 24 hours, and assayed forluciferase activity.

In other cases, Huh-7.5 cells are infected with DENV (multiplicity ofinfection=1) for 4 hours and then treated with test compounds at varyingconcentrations. Twenty four hours post-infection, viral RNA levels orreleased virus are quantified along with cellular ATP levels.

Three types of in vitro assays for DENV include those described in Chenet al., Antimicrobial Agents and Chemotherapy, 2010, 54(8):3255-3261.

Type 1: The assays measure viral titer reduction in the presence of testcompounds. Vero cells are seeded in a 12-well plate (4×10⁵ cells perwell). At 24 hours post-seeding, the cells are infected with DENV at amultiplicity of infection of 0.1 and treated immediately with the testcompounds. Culture medium are collected at an appropriate time, andviral titers are determined using plaque assays.

Type 2: Cell-based flavivirus immunodetection (CFI) is used to measurethe amount of viral E protein in infected cells. A549 cells are seededto a 96-well plate (2×10⁴ cells per well). The cells are infected withDENV on the following day. During the infection, the cells are incubatedwith a test compound/virus mixture for 1 hour, with shaking every 10 to15 minutes. The culture fluid is then replenished with fresh mediumcontaining test compounds. On day 2 post-infection, the cells are washedwith PBS, fixed with 100% methanol at 4° C. for 10 minutes, and detectedfor intracellular viral E protein by ELISA. The ELISA uses mousemonoclonal antibody 4G2 against the DENV E protein and goat anti-mouseIgG conjugated with hoseradish peroxidase as the primary and secondaryantibodies, respectively.

Type 3: The assay uses Huh-7 cells and luciferase-reporting replicon ofDENV. The procedures are similar to those described above.

6.7 In Vivo Assays

6.7.1 In Vivo Assays for Herpes Viruses

6.7.1.1 HSV-1 and HSV-2

Animal Models of Herpes Encephalitis:

Virus Species Route Disease HSV-1 BALB/c Mice i.p. Encephalitis i.n.Encephalitis HSV-2 BALB/c Mice i.p. Encephalitis i.n. Disseminatedinfection of newborns and encephalitis HSV-1 Rat i.n. Encephalitis HSV-1SKH-1 Mice i.cut. Herpes labialis

New compounds are screened initially for HSV activity in BALB/c mice(Charles River Laboratories) inoculated i.p. with HSV-1 or HSV-2.Following i.p. inoculation with HSV-1 or HSV-2, virus replicates in thegut, liver, and spleen and spreads to the CNS by viremia and likelyperipheral nerves as well. Virus is detected first in the brain aroundday five, thus allowing time for compounds to demonstrate anti-viraleffects. This model system one of the most sensitive for determiningefficacy of a new anti-viral compound. Although it does not simulate anatural route of infection, it allows for screening new compounds todetermine optimal dosages and treatment regimens. This screening isfollowed by testing in mice inoculated i.n. which more closely simulateshuman infections. If the experimental compound exhibits activity in miceinoculated i.p., it is next evaluated in mice inoculated by the i.n.route.

I.n. inoculation of three-week-old BALB/c mice with HSV-1 provides amodel for herpes encephalitis of humans utilizing a natural route ofinfection. After inoculation of approximately 105 pfu of HSV-1, strainE-377, virus replicates in the nasopharynx and spreads to the CNS by wayof olfactory and trigeminal nerves. Untreated animals generally die bydays 6-10. The use of i.n. inoculation is known as a natural route ofinfection for herpes encephalitis. I.n. inoculation of three-week-oldBALB/c mice with about 4×10⁴ pfu of HSV-2, strain MS, provides a modelof disseminated neonatal herpes with CNS involvement. After viralinoculation, virus replicates in nasopharyngeal and lung tissue, anddisseminates to the liver, spleen, and kidney. In addition, virusspreads to the CNS via olfactory and trigeminal nerves. Acyclovir, ACV,given parenterally or orally is effective in all the experimentalinfections mentioned above and is utilized as a positive control.

The SKH-1 strain of immunocompetent hairless mice is used to facilitatescoring of cutaneous lesions. Orofacial inoculation of HSV-1 in thesemice provides an appropriate model for testing new anti-viral therapies.In this model, mice are anesthetized with a ketamine/xylazine mixtureand injected subcutaneously with an electronic microchip for individualidentification. Prior to inoculation, the snout, composed of thetriangular shaped area over the nasal bones from the nose bridge to theeyes, is lightly abraded with a #113 tungsten-carbide engraving bitDremmel tool. This procedure is performed carefully to prevent bleeding.This area is then swabbed for 10 seconds with a dacron swab soaked withHSV-1. Following this procedure, animals are returned to their cages andobserved until recovery.

Animals infected with HSV-1 in the orofacial area exhibit lesions thatbegin to appear on days 4-6 and are usually cleared by day 15. Todetermine the effect of treatment on cutaneous viral replication,severity of lesions is scored from days 4-21 and swabs of the snout areaare taken on days 3-10. The samples are placed in 2.0 mls of media andfrozen at −70° C. until titrated for HSV-1 on rabbit kidney fibroblastcells in a CPE microtiter plate assay. All experimental drug efficacystudies are placebo or vehicle controlled and also the positive control,Zovirax, is administered topically.

Mouse Model of Primary HSV-1/HSV-2 Challenge:

The primary screening model provides a rapid initial evaluation ofanti-viral efficacy against HSV primary infection with both clinical andvirological endpoints. This model utilizes intravaginal inoculation offemale Swiss Webster mice (25 g) with HSV-1 or HSV-2 to evaluatepotential anti-viral therapies as well as vaccine/adjuvant candidates.Animals are followed daily for signs and systems of herpes disease andvaginal swabs are obtained to evaluate the effect of therapy on viralreplication. Single or combined anti-viral therapies can be administeredtopically, orally or systemically and can be given at varying intervalsbegun before or after viral challenge. Dose range studies can also beperformed. Dose and route of administration are individualized for eachexperimental compound. Treatment group size is typically 12-16 mice.

Microbicide Screening Model of Primary HSV-2 Challenge in Mice:

This model is designed to evaluate the protection afforded by amicrobicide candidate against infection with HSV-2. The model utilizesintravaginal inoculation of female Swiss Webster mice for evaluation.The initial trial is usually performed by applying compound 5 minutesprior to challenge with HSV-2. Further evaluation of microbicides inthis model either extend the time between microbicide administration andchallenge or examine dose range. Compounds can be advanced to asecondary species evaluation in the guinea pig model of genitalinfection. Evaluation includes daily evaluation for signs and symptomsof genital herpes and viral examination of vaginal secretions. Treatmentgroup size is typically 12-16 mice.

Guinea Pig Model of Primary Genital HSV-2 Infection:

Because genital herpes disease in the guinea pig more closely resembleshuman disease, this animal is used as a second species for therapieswith demonstrated efficacy against HSV in mice. As with humans, genitalHSV infection in guinea pigs is a self limited vesiculoulcerativedisease which is followed by healing, the establishment of latency, andthen both spontaneous and inducible symptomatic and asymptomaticrecurrences. An exemplary model utilizes intravaginal inoculation offemale Hartley guinea pigs and provides both clinical and virologicindices to asses both the effect of treatment on primary disease as wellas on the frequency or severity of subsequent recurrent infections.Anti-viral therapy can be administered orally, topically or systemicallyand can be given at varying intervals beginning before or after viruschallenge. Following intravaginal inoculation, animals are followeddaily for the development of genital herpes using a validated genitalherpes scoring system. Vaginal swabs are also obtained to evaluate theeffect against viral replication. Because this is a non lethal model,animals can be sacrificed at the conclusion of the experiment toevaluate the effects of treatment on latency. This model can be adaptedto evaluate anti-viral activity against available drug resistant strains(ACV and Foscarnet). Dose, route of administration and duration oftreatment are individualized for each experimental agent. Treatmentgroup size is typically 10-15 animals.

Guinea Pig Model of Recurrent Genital HSV-2 Infection:

The guinea pig model of genital herpes is unique in that after recoveryfrom primary genital infection, animals experience spontaneous recurrentgenital lesions as well as viral shedding in the absence of lesions.This allows a candidate compound to be evaluated for efficacy incontrolling recurrent disease. Female Hartley guinea pigs who haverecovered from symptomatic primary genital infection are randomized intotreatment groups for anti-viral treatments beginning on day 21 PI andcontinued for 21 days after. Treatments can be given orally, topicallyor systemically. The indices for these studies include quantificationand severity assessment of recurrent episodes during treatment and for21 days following cessation of treatment. Additionally, vaginal swabsare collected to evaluate any impact on shedding. Dose, route ofadministration are individualized for each experimental agent. Treatmentgroup size is typically 10-15 animals, and duration of treatment istypically 21 days.

Model of Neonatal HSV-2 Infection in Guinea Pigs:

An exemplary model of neonatal HSV infection mimics the natural historyof infection in the human newborn. This model is available to evaluatecandidate anti-viral compounds and combined therapeutic approachesincluding combination of anti-virals or anti-virals and immunemodulators. Additionally, this model can be used to evaluate theefficacy of candidate vaccines by measuring the protection afforded bytransplacental antibody. In this model, newborn Hartley guinea pigs areinoculated intranasally with HSV-2 within 48 hours of delivery. Newbornanimals are then randomized to receive experimental compound, placebo orACV (control). A positive control of ACV (60 mg/kg/day) BID is typicallyused. Animals are evaluated daily for evidence of cutaneous herpeticdisease and weight gain as well as pulmonary, CNS symptoms and death.Surviving animal are followed for 45 days to assess the effectiveness oftherapy on the incidence and frequency of cutaneous herpeticrecurrences. Dose and route of administration are individualized foreach experimental agent. Duration of treatment is typically 10 days ormore.

6.7.1.2 Cytomegaloviruses

Animal Models for Cvtomegalovirus Infections:

Virus Species Route Disease MCMV BALB/c mice i.p. Disseminated CMVacute, chronic SCID-mice i.p. Disseminated CMV acute HCMV SCID-hu-Reti.oc. HCMV replication in Retinal tissue SCID-hu-thy/liv i.im. HCMVreplication in thymus/liver tissue

Human CMV does not generally infect laboratory animals. For this reason,it is necessary to use surrogate models, that is, a similar butdifferent virus in its natural host. While there are cytomegalovirusstrains in a number of animal species, two that have been studiedinclude the murine and guinea pig CMVs. The murine model is predictiveof efficacy for anti-viral drugs, such as Foscarnet (PFA), ganciclovir(GCV), and cidofovir (CDV) that have been evaluated in humans.

I.p. inoculation of three-week-old BALB/c mice with approximately2.0×10⁵ pfu of MCMV results in an acute, lethal infection with rapidvirus replication in the lung, liver, spleen, kidney, intestine,salivary gland, and other visceral and glandular tissue. Animals die onapproximately days 5-7. Since this is a lethal infection, the model canbe used for rapid identification of potential anti-viral compounds.Reduction of the virus inoculum to 10⁴ pfu of MCMV results in anon-lethal, chronic, generalized infection, which has many similaritiesto human CMV infections. At various times after inoculation, virus canbe readily isolated from blood, lung, liver, spleen, kidney, urine,intestine, and salivary gland. Virus replication persists in thesetarget organs for 45-60 days and in the salivary gland for months. Thenature of the chronic infection allows for evaluating long term ormaintenance therapy.

Severe combined immunodeficient (SCID) mice, which lack functional T andB cells, are extremely sensitive to infection with MCMV and are utilizedas models for CMV infections in an immunocompromised host. SCID micethat are inoculated with a range of 1.0-10⁵ pfu of MCMV, and are leftuntreated, eventually die in a dose dependent manner. Animals thatreceive 10⁵ pfu have a mean day of death of about 14 days, whereas,those inoculated with 10 pfu survive an average of 25 days. With eachlog 10 increase in virus inoculum, survival time is decreased by aboutthree days. To determine the pathogenesis of MCMV in SCID mice, mice areinoculated with 10 pfu. On each of various days post infection, threemice are euthanized, their tissues removed, homogenized, and assayed forMCMV. Virus is first detected in salivary gland by day six followed bylung, spleen, kidney, adrenals, and pancreas on days 9-12. Liver, whichis one of the most permissive organs in normal mice, does not exhibitdetectable virus until day 18. In addition, brain is infected by day 18.These data indicate that inoculation of SCID mice with lowconcentrations of MCMV results in a disseminated infection with viralreplication in the same target organs as observed in immunodeficienthumans. These animals demonstrate high levels of virus in their tissuesfor 2-3 weeks, thus allowing adequate time to document an anti-viralresponse in treated animals compared with placebo animals.

Human CMV infections can cause a wide range of clinical manifestations,especially in the immunocompromised host. Few models exist to study HCMVinfection since the virus is host-specific, and infection andreplication are limited to human cells. In this regard, a model thatinvolves HCMV infection of fetal human retinal tissue implanted in theeyes of severe combined immunodeficient (SCID) mice can be utilized.Small fragments of fetal human retinas are implanted into the anteriorchamber, and four to six weeks after transplantation are inoculated with2,000 to 10,000 pfu of HCMV. Animals are euthanized and eyes enucleatedat various time points after infection. Eyes are prepared for microscopyby sectioning fixed tissue, or are homogenized for detection ofinfectious HCMV by plaque assay. The model has been validated using GCV,CDV, and other anti-viral therapies. In addition, this model can also beutilized to study and identify the virulence characteristics of HCMV byexamining the growth of various HCMV mutants.

The SCID-hu thy/liv implant model can also be used in compound efficacystudies. In this model, small fragments of human fetal thymus and liverare implanted under the kidney capsule in the SCID mouse. Approximately12-16 weeks later, implants that are fully vascularized and quite large(10-50% the size of the kidney) are inoculated with 10³-10⁴ pfu of HCMV.At various time points after infection, implants are biopsied andhomogenized, and HCMV replication is quantified by plaque assay. As withthe SCID-hu mouse ocular model, this model can be useful in determiningthe efficacy of various anti-viral therapies.

Immunocompromised GPCMV Model:

This guinea pig model mimics CMV infection of the immunocompromisedhost, a common target population of cytomegalovirus infections. YoungHartley guinea pigs are immunossuppressed with cylcophosphamideadministered 1 and 7 days prior to viral inoculation with ˜10⁵ pfusalivary gland passaged guinea pig cytomegalovirus (GPCMV). In a typicalexperiment, two groups of 12 animals each receive the experimentalcompound or placebo beginning 24 hours after infection. Animals arefollowed daily for evidence of disease and death which usually occurs byday 14 as described in Bourne et al., Antiviral Research 2000,47:103-09. Effects on viral replication are measured by sacrificinganimals and quantitating virus in specific organs and blood by Real-TimePCR and/or culture. The amount of compound is typically based on anaverage guinea pig weight of 350-500 g.

Neonatal GPCMV Model:

CMV infection of premature newborns may be life-threatening disease ifuntreated. The neonatal guinea pig model resembles perinatal CMVinfection and allows systematic evaluation of anti-viral compounds in arelatively immature host. In this model, newborn Hartley guinea pigs areinfected with ˜10⁶ pfu of salivary gland derived GPCMV 24-48 hours afterbirth. Anti-viral or placebo treatments, administered orally or byintraperitoneal injection are begun at 0-24 hours after infection.Infection results in decreased weight gain and mortality as high as 70%due to dissemination to target organs such as the liver, spleen andbrain by day 10 post-infection (Bravo et al., Antiviral Research 2003,60:41-49). Animals are followed daily for signs of disease and death.The effects on viral replication are assessed by sacrificing animals andcomparing viral titers in various target organs and blood by Real-TimePCR and/or culture. Dosing is typically based on an average newbornguinea pig weight of 100 g.

Congenital GPCMV Model:

CMV is the most common congenital infection. The guinea pig is a smallmammal in which virus crosses the placenta to cause fetal infection anddisease, thus allowing the study of new anti-virals and unique therapiesthat may target placental and congenital infection. In this model,Hartley pregnant guinea pigs are infected with ˜10⁵ pfu GPCMV atapproximately 45 to 55 days of a 70 day gestation. Animals can betreated by systemic or oral routes. Endpoints include prevention ofpremature delivery, survival of the offspring and PCR analysis ofplacenta, and other maternal tissues (blood, liver and spleen) and puporgans (liver and spleen) harvested 3, 5 or 10 days post infection(Bravo et al., Journal of Infectious Diseases 2006, 193:591-7). The doseis typically based on a pregnant guinea pig weight of about 1200 g.

CMV Model of Hearing Loss:

Hearing loss is the most common manifestation of congenital CMVinfection. Using direct inoculation of GPCMV (˜10⁵ pfu) into the cochleathrough the round window, hearing loss can be induced in guinea pigs asmeasured by ABR. Animals can then be treated to prevent the hearingloss. Test compounds can be administered either systemically, orally andpossibly direct intratympanic administration. Dose is typically based onthe weight of the animals, approximately 350 g.

Murine CMV Model:

The murine CMV model is used to study CMV pathogenesis and to evaluatenew anti-CMV compounds. In this model, 5-week old female mice areinfected with 1×10⁶ pfu of MCMV by intraperitoneal injection. Treatmentcan begin before or following infection and lasts 3-5 days. Animals aresacrificed at 3 to 5 days after infection and viral titers of the spleenand liver are determined by plaque assay. Other tissues such as salivarygland and lungs can be analyzed as well. Ganciclovir (50 mg/kg, twicedaily) serves as a control drug and inhibits MCMV replication in thismodel. Dosing depends on the weight of the animals, typically about 25g.

6.7.2 In Vivo Assays for Influenza Viruses

Efficacy: The influenza animal model consists of an infection oflaboratory mice with various strains of influenza A (H1N1, H3N2, H5N1)and B viruses, with the employment of several parameters to measuredisease severity. The parameters which can be used include thefollowing: (a) monitoring of blood oxygen saturation (SaO₂) levels inlive animals at frequent intervals utilizing pulse oximetry; (b)measuring of infectious pulmonary virus titers using in vitro endpointdilution assay of homogenates of lungs taken at designated intervalsduring the infection; (c) assay of the degree of pulmonary consolidationusing lungs taken in as determined both by score of lung discolorationand by weight of the lung; (d) death of the animal due to viralpneumonia; (e) mean survival time of the animals; and (f) selectedhistopathological analysis of lung sections. Where appropriate, studiesare conducted to determine the development of viruses resistant tosignificant anti-viral drugs.

Toxocity:

One or more toxicity determinations are performed on the test compoundsunder evaluation. These determinations include: (a) lethality; and (b)host weight loss or failure to gain weight. As needed and whereapplicable, the following additional parameters can also beinvestigated: (a) increase in circulating serum levels of glutamicoxalic acid transaminase (SGOT) and pyruvic acid transaminase (SGPT) inthe serum as markers for possible liver damage; (b) increase incirculating creatinine (CT) level as an indicator of possible renalimpairment; and (c) increase in circulating creatinine phosphokinase(CK) levels as indicator of general tissue damage.

6.7.3 In Vivo Assays for Respiratory Viruses

6.7.3.1 RSV, PIV-3, MV and hMPV

Respiratory syncytial virus (RSV), parainfluenza virus type 3 (PIV-3),measles virus (MV) and human metapneumovirus (hMPV) are human pathogenswhere there is a lack of licensed vaccines for preventing illnessescaused by RSV, PIV-3, or hMPV, although efficacious MV vaccines areavailable. Ribavirin, immune serum globulins and the humanizedmonoclonal antibody have been approved for use against some of theseparamyxoviruses. However, all of these agents have limitations and canbe expensive. Thus, the elucidation and development of new compounds,reagents or vaccines with activity against these viruses are needed.Potential anti-virals and vaccines that can be effective against RSV,PIV-3, MV or hMPV are evaluated in cotton rats. In addition, studies areperformed to characterize, enhance or further develop the differentparamyxovirus-cotton rat models. Evidence obtained in numerous studiessupport the usefulness of the different paramyxovirus-cotton rat modelsfor preclinical evaluation of potential paramyxovirus anti-virals andvaccines.

6.7.3.2 SARS Virus

Efficacy:

The SARS virus animal model utilizes weanling mice infected intranasallywith the virus. A moderate lung infection is manifested by occasionallung hemorrhaging but primarily by infectious virus recovered from thelungs. Inhibition of development of virus in the lungs of the mice isused as parameters for evaluation of test agents.

Toxicity:

One or more toxicity determinations are performed on the test compoundsunder evaluation. These determinations are: (a) lethality; and (b) hostweight loss or failure to gain weight. As needed and where applicable,the following additional parameters can also be investigated: (a)increase in circulating serum levels of glutamic oxalic acidtransaminase (SGOT) and pyruvic acid transaminase (SGPT) in the serum asmarkers for possible liver damage; (b) increase in circulatingcreatinine (CT) level as indicator of possible renal impairment; and (c)increase in circulating creatinine phosphokinase (CK) levels asindicator of general tissue damage.

6.7.4 In Vivo Assays for Orthopoxviruses

6.7.4.1 Vaccinia and Cowpox Viruses (Smallpox Assay)

The smallpox animal model is an intranasal infection of laboratory miceby the cowpox and vaccinia viruses, which induce an infection of thenose and lungs resulting in a smallpox-like toxemia-associated death.Parameters used in evaluating test compounds in this model include: (a)death of the animal; (b) mean survival time of the animals; (c) lung andnose virus titers; and (d) host weight loss. Other parameters caninclude: (a) monitoring SaO₂ levels; (b) assay of degree of pulmonaryconsolidation both by lung score and lung weight increase; and (c)histopathological analysis of lungs and other organs.

Also utilized is a cutaneous infection in immunocompromised hairlessmice that can be induced by vaccinia virus. This infection isprogressive and leads to the death of the mice. It is now also beingused in selected anti-viral experiments. Parameters used in evaluatingtest agents in this cutaneous infection model include: (a) death of theanimal; (b) severity score in initially induced lesions; (c) size ofinitially induced lesions; (d) number of spontaneous “satellite”lesions; and (e) virus titer in various organs in the animal.

Animal Models for Vaccinia and Cowpox Virus Infections:

Virus Species Route Disease Cowpox Virus (BR) BALB/c Mice i.p. Death -Rapid Liver-Visceral Involvement BALB/c Mice i.n. Death - SlowerLung-respiratory involvement Vaccinia Virus (WR) SKH-1 mice i.d. Skinlesions SCID Mice i.p. Disseminated disease BALB/c Mice i.n. DeathDisseminated Disease Vaccinia Virus (IHD) BALB/c Mice i.n. DeathDisseminated Disease Vaccinia Virus (WR) SCID Mice i.p. DeathDisseminated Disease Vaccinia Virus (NYC) SCID Mice i.p. Death

The causative agent of smallpox, variola virus, cannot be utilizedoutside a BSL-4 containment area and does not cause disease in adultmice. Various orthopoxviruses can be utilized as surrogate viruses forsmallpox including VV and CV. They can be inoculated i.p. or i.n. intoSCID mice with an endpoint of death. In normal mice, CV, VV-WR, orVV-IHD, but not VV-Copenhagen Strain, will produce mortality wheninoculated by variety of routes. Intranasal inoculation of mice with CVproduced an infection with features similar to systemic or disseminatedsmallpox. Other routes of inoculation such as i.p. or i.v. with VV or CVresult in less bronchial involvement and more skin lesions. The IHDstrain of VV is less virulent in BALB/c mice than the WR strain. The WRstrain of VV produces mortality in BALB/c mice by i.n. inoculation andSCID mice by i.p. inoculation. SKH-1 hairless mice can also beinoculated with VV and CV by inoculation of abraded orofacial areas,similar to the HSV techniques. Mice can be treated systemically ortopically with anti-viral compounds for evaluation of efficacy againstdisease (lesion scores) or viral replication (viral titers).

6.7.4.2 Ectromelia (Mousepox Assay)

Ectromelia virus is the causative agent of mousepox, an acuteexanthematous disease of mouse colonies in Europe, Japan, China, and theUSA. Laboratory studies have shown ECTV to have a very narrow hostrange, infecting only certain mouse species. A number of differentstrains of ECTV have been isolated which have been shown to differ intheir virulence for the mouse. The Moscow, Hampstead, and NIH79 strainshave been studied, with the Moscow strain being one of the mostinfectious and virulent for the mouse. Studies in the last five decadeshave resulted in a detailed description of the virologic and pathologicdisease course in genetically susceptible (A, BALB/c, DBA/2, and C3H/He;death ˜7 days post-infection) and resistant (C57BL/6 and AKR) inbred andout-bred mice; identification and characterization of the importantcell-mediated and innate responses for recovery from infection; and thediscovery of rmp-1, rmp-2, rmp-3 and rmp-4 loci which govern resistanceto severe mousepox. Varying mouse genotype, virus strain, and dose ofvirus result in distinct disease patterns for a given route ofinfection.

Mousepox differs from smallpox in at least two features following arespiratory tract infection. First, the disease course in mousepox isshorter as compared to smallpox. The eclipse period in mousepox andsmallpox are 6 and 10 days, respectively. Fatal cases of mousepoxusually occur 7 to 14 days post-infection (p.i.), whereas deaths inordinary smallpox occur from ˜18 to 22 days p.i. Second, the majorlesions in mousepox are observed in the liver and spleen, whereas theseorgans are relatively uninvolved in smallpox. A feature of mousepox thatis similar to smallpox is the relatively small dose of virus that isrequired to initiate disease in the upper and lower respiratory tract.Another similarity is the detection of virus in respiratory gases duringthe preexanthem period. Additionally, both diseases present with acharacteristic exanthematous rash. In the case of mousepox, thedevelopment of rash is dependent on a number of parameters includingmouse strain, virus strain, route of inoculation and virus dose.

Efficacy:

An important use of the mousepox model is the evaluation oforthopoxvirus compounds and vaccines. The ECTV aerosol model provides abroad dynamic range for evaluating compounds. An aerosol lethal dose of100 PFU can be used, which is ˜3-fold greater that the LD₅₀ value of 32PFU, and is likely in the range of the infectious dose for aerosolizedsmallpox. Alternatively, a dose 1000 to 10,000 times the LD₅₀ can beused to fully examine the robustness of the test compound.

6.7.4.3 Monkeypox Virus (MPXV)

Animal assays for monkeypox virus (MPXV) can be performed by followingthe procedures described in, e.g., Americo et al., Journal of Virology,2010, 84(16): 8172-8180. Generally, the assay is based on an intranasalor intraperitonial infection of CAST/EiJ mice with MPXV, for example, anisolate of Congo Basin clade of MPXV or West African clade of MPXV. Uponinfection, the animals exhibit loss of weight, morbidity and death in adose dependent manner. In addition, MPX replication is observed in thelung, spleen and liver of the tested animals.

Consequently, anti-viral efficacy of the test compounds can be assessedby following the criteria such as weight loss, morbidity and death inthe presence and absence of the test compounds upon infection with MPXV.Further, replication in organs such as lung, spleen and liver of theanimals in the presence and absence of the test compounds upon infectioncan also be examined to assess the anti-viral efficacy.

6.7.4.4 Rabbitpox Virus (RPV)

Animal assays for rabbitpox virus (RPV) can be performed by followingthe procedures described in, e.g., Rice et al., Viruses, 2011, 3:63-82,and Adams et al., J. Virol., 2007, 81:11084-11095 ( ). Generally, themodel is based on bilateral, intrademal infection of New Zealand Whiterabbits with 100-1000 pfu of RPV. Upon infection, the animals exhibitweight loss, elevated body temperature (fever), severe respiratorydistress, swelling of primary and secondary lesions, eye and nasaldischarge and inoculation site necrosis. In addition, viral replicationis observed in the respiratory tract. If untreated, the animals areeventually subjected to death (euthanasia) according to euthanasiaguidelines.

Consequently, anti-viral efficacy of the test compounds can be assessedby following the criteria described above, including length of survivalupon injection and viral replication. In addition, an overall clinicalscore can be examined to assess the anti-viral efficacy.

Alternatively, animal assays can be based on the procedures described,e.g., in Roy et al., Viruses, 2010, 2:2096-2107, in which similarclinical criteria are examined following an infection through aerosolcontaining RPV. In this model, experimental infection with RPV initiateswith exposure to aerosols with a particle size distribution that ispreferential for penetration to the tracheobronchial and pulmonaryregions of the lung, with emphasis on the lower respiratory tract.

6.7.5 In Vivo Assays for Papillomaviruses

6.7.5.1 Cottontail Rabbit Papillomavirus (CRPV) Model

The procedures substantially similar to those described, for example, inChristensen, Antiviral Chemistry & Chemotherapy, 2005, 16:283-294 arefollowed in connection with cottontail rabbit papillomavirus (CRPV)model. In short, topical formulations of the test compound are tested atthree doses in groups of 5 rabbits at 4 sites per rabbit. One additionalrabbit group includes a placebo treatment. Alternative deliveriesinclude interalesional and systemic treatments depending on the natureof the compound to be tested (e.g., anti-viral, immunomodulator).

Adult New Zealand White rabbits can be purchased from, for example,CoVance, Inc. Rabits are of both genders. Rabbits are quarantined andcleared (14 days). Each rabbit is inoculated with 10⁻² wtCRPV (4 sites:2 on the left side of the back (L1 and L2) and 2 on the right side ofthe back (R1 and R2)) CRPV stock. Combinations of L1, R1, L2 and R2sites receive treatments. Exemplary treatment schemes are providedbelow.

-   -   Group A: all 4 sites=placebo ointment;    -   Group B: L1 and L2=GS327422 (0.1%); R1 and R2=GS327422 (0.03%)        Treatments are once/week (Monday) for 8 weeks;    -   Group C: L1 and L2=GS327422 (0.1%); R1 and R2=GS327422 (0.03%)        Treatments are three times/week (MWF) for 8 weeks; and    -   Group D: L1 and L2=GS327422 (0.1%); R1 and R2=GS327422 (0.03%)        Treatments are five times/week (MTWTF) for 8 weeks.

The experiment contains 20 rabbits. Most experiments include 4-5 groupsof rabbits (Groups A-E). A placebo group serves as controls to assesslocal effects of treatment in treated Groups B to D. Vehicle consists ofplacebo. Groups B-D represents test compound comparisons vs placebonegative control. Doses of compounds are chosen based on variouscriteria including the past experience. Treatments (topical) begin at atime when the papillomas are visible but not greater than a GMD of 5.0mm. This time point allows effects on visible papillomas to be assessed,and is a clinically relevant situation. Treatment is once weekly (GroupB), 3 times weekly (group C—MWF), and 5 times weekly (MTWTF), for eightweeks with a dose of 0.1 ml per site. Alternatively, treatments maybegin 14 days after infection at a time when there are no visiblepapillomas to maximize the effectiveness of the treatments. Body weightsare taken weekly, and sera collected at the end of the treatment periodfor blood chemistries as needed. Papillomas are measured weekly in 3axes (length×width×height) in mm. Data are entered into a spread sheetand calculations conducted of the geometric mean diameter of eachpapilloma, mean±SEM for each group, t-test between each paired groupsand plots made of papilloma size vs time. Plots of weight changes arealso conducted. At termination, kidney and liver samples are retrievedfor histology and toxicity assessment. Skin/papilloma sites aremonitored photographically and biopsies assessed for histology atexperiment/treatment termination. Serum samples can be collected toconduct blood chemistries to assess any toxicities of the compound undertreatment.

6.7.5.2 Mouse Xenograft Model

Subcutaneous and cutaneous mouse xenograft models are schematicallydescribed in FIGS. 1 and 2.

6.7.6 In Vivo Assays for Other Viruses

6.7.6.1 Punta Toro Virus

Efficacy:

The Punta Toro virus infection is achieved in C57BL/6 mice and in Syriangolden hamsters, with a generalized disease resembling that induced byRift Valley fever. Parameters used for anti-viral testing include: (a)death of the animal; (b) hepatic icterus, seen as yellowed liver; (c)elevated ALT levels in serum; (d) virus titers in liver and serum; and(e) host weight loss.

Toxicity:

One or more toxicity determinations are performed on the test compoundsunder evaluation. These determinations are: (a) lethality; and (b) hostweight loss or failure to gain weight. As needed and where applicable,the following additional parameters can also be ivnestigated: (a)increase in circulating serum levels of glutamic oxalic acidtransaminase (SGOT) and pyruvic acid transaminase (SGPT) in the serum asmarkers for possible liver damage; (b) increase in circulatingcreatinine (CT) level as indicator of possible renal impairment; and (c)increase in circulating creatinine phosphokinase (CK) levels asindicator of general tissue damage.

6.7.6.2 Pichinde Virus

Efficacy:

The Pichinde virus model utilizes Syrian golden hamsters. Parametersused for anti-viral testing include: (a) death of the animal; (b) virustiters in brain, liver, spleen and serum; and (c) elevated ALT levels inserum.

Toxicity:

One or more toxicity determinations are performed on the test substancesunder evaluation. These determinations are: (a) lethality; and (b) hostweight loss or failure to gain weight. As needed and where applicable,the following additional parameters can also be investigated: (a)increase in circulating serum levels of glutamic oxalic acidtransaminase (SGOT) and pyruvic acid transaminase (SGPT) in the serum asmarkers for possible liver damage; (b) increase in circulatingcreatinine (CT) level as indicator of possible renal impairment; and (c)increase in circulating creatinine phosphokinase (CK) levels asindicator of general tissue damage.

6.7.6.3 VEE Virus

Efficacy: The VEE virus animal model utilizes the TC-83 vaccine strainof virus administered intranasally to C3H/Hen mice; the virus progressesto the central nervous system causing high virus titers in the brain anddeath of the animal. The Semliki Forest virus model is very similar tothat for the Banzi virus, with the same disease parameters. The SemlikiForest virus is a BSL-3-rated pathogen which requires special handling.Parameters for evaluation include: (a) death of the animal; (b)prolongation in mean day to death; (c) virus titers in the brains; and(d) host weight loss.

Toxicity:

One or more toxicity determinations are performed on the test substancesunder evaluation. These determinations are: (a) lethality; and (b) hostweight loss or failure to gain weight. As needed and where applicable,the following additional parameters can also be ivnestigated: (a)increase in circulating serum levels of glutamic oxalic acidtransaminase (SGOT) and pyruvic acid transaminase (SGPT) in the serum asmarkers for possible liver damage; (b) increase in circulatingcreatinine (CT) level as indicator of possible renal impairment; and (c)increase in circulating creatinine phosphokinase (CK) levels asindicator of general tissue damage.

6.7.6.4 West Nile Virus

Efficacy:

The West Nile virus animal model currently utilizes both mice andhamsters. In each, neurological signs are produced, leading to eventualdeath of the animals. This virus is a BSL-3-rated pathogen which isrecovered from various tissues. Other parameters such as functionalabilities are also reviewed. Disease parameters used for anti-viralevaluation include: (a) death of the animal; (b) prolongation on meanday to death; (c) virus titers in brain and other tissues; and (d) hostweight loss.

Toxicity:

One or more toxicity determinations are performed on the test substancesunder evaluation. These determinations are: (a) lethality; and (b) hostweight loss or failure to gain weight. As needed and where applicable,the following additional parameters can also be ivnestigated: (a)increase in circulating serum levels of glutamic oxalic acidtransaminase (SGOT) and pyruvic acid transaminase (SGPT) in the serum asmarkers for possible liver damage; (b) increase in circulatingcreatinine (CT) level as indicator of possible renal impairment; and (c)increase in circulating creatinine phosphokinase (CK) levels asindicator of general tissue damage.

6.7.6.5 Dengue Virus

In vivo assays for DENV can be conducted using procedures substantiallysimilar to those described, for example, in Guabiraba et al., PLoS ONE,2010, 5(12):e15680 and Souza et al., Proc. Natl. Acad. Sci., 2009,106(33):14138-14143. DENV virus stock solutions are diluted inendotoxin-free PBS or DPBS to appropriate concentrations. The virus isinjected i.p. into mice. Test compounds are given via appropriate routesat appropriate dosing frequency (e.g., twice a day oral administration).Lethality rates are evaluated every 12 hours and other parameters (bodyweight loss, inflammation, etc.) are checked as appropriate. For testsusing knock-out mice, the control typically includes the same test onthe wild-type mice. Negative control usually involves the administrationof vehicle instead of test compound.

In the case of evaluation of vaccines for DENV virus, assays can beconducted using procedures similar to those described, for example, inJohnson et al., Journal of Virology, 1999, 73(1):783-786. The assay usesIFN deficient mice (e.g., A129 mice, which lack alpha/bea IFN and gammaIFN receptor genes) and involves intraperitoneal administration of DENVinto such mice. Typically, IFN deficient mice are universally lethalupon administration of DENV regardless of age. Based on this, criteriasuch as changes in survival time and rate can be monitored in IFNdeficient mice immunized with test vaccine to assess the efficacy of atest vaccine in vivo.

6.7.7 In Vivo Assays for Prion Diseases

Efficacy:

The prion transgenic mouse model utilizes knockout mice for endogenousmouse PrP-sen. These mice express high levels of hamster PrP-sen in awide range of tissues, including the brain. The animals infected withhamster scrapie agent replace the Syrian hamster model. The latteranimals require approximately 120 days to die of the scrapie infection,whereas the prion transgenic mice die in approximately 82 days wheninfected with the same agent. Death is used as the parameter foranti-prion evaluation.

Toxicity:

One or more toxicity determinations are performed on the test substancesunder evaluation. These determinations are: (a) lethality; and (b) hostweight loss or failure to gain weight. As needed and where applicable,the following additional parameters can also be ivnestigated: (a)increase in circulating serum levels of glutamic oxalic acidtransaminase (SGOT) and pyruvic acid transaminase (SGPT) in the serum asmarkers for possible liver damage; (b) increase in circulatingcreatinine (CT) level as indicator of possible renal impairment; and (c)increase in circulating creatinine phosphokinase (CK) levels asindicator of general tissue damage.

6.7.8 Other Follow-Up Tests

Follow-up determinations of promising anti-virals seen in the originalanimal studies can include effect of the administered test compounds onkey immunologic components in infected and in uninfected (toxicitycontrol) mice. The immunologic effects studied include: (a) cytotoxic Tlymphocyte activity; (b) natural killer cell activity; (c) total T,T-helper, T-suppressor/cytotoxic and B cell enymeration; (d) response tothe T-cell mitogen phytohemagglutinin (PHA); (e) production ofinterferon; and (f) production of neutralizing antibody. Whereappropriate, studies are conducted to determine the development ofviruses resistant to significant anti-viral drugs.

7. Assays for ELOVL

ELOVL assays can be conducted in vitro using procedures substantiallysimilar to those described in, for example, Shimamura et al., EuropeanJournal of Pharmacology, 2010, 630: 34-41.

7.1 In Vitro Assays

7.1.1 Elongation Enzyme Assay

Elongation is carried out using 30 μl of substrate reaction mixturecontaining 100 mM potassium phosphate buffer (pH 6.5), 200 μM BSA (fattyacid free), 500 μM NADPH, 1 μM rotenone, 20 μM malonyl-CoA, 833 kBq/ml[¹⁴C]malonyl-CoA (GH Healthcare Science, Little Chalfont, UK) andacyl-CoA. The following long chain acyl-CoAs are used as a preferentialsubstrate for each ELOVL: ELOVL1, 10 μM stearoyl-CoA; ELOVL2, 10 μMarachidonoyl-CoA; ELOVL3, 10 μM stearoyl-CoA; ELOVL5, 40 μMarachidonoyl-CoA; and ELOVL6, 40 μM palmitoyl-CoA. To start thereaction, 20 μl of the ELOVL microsomal fraction is added to thesubstrate mixture, and then incubated for 1 hour at 37° C. with gentleshaking in a 96-well plate. After 1 h incubation, 100 μl of 5 M HCl isadded to hydrolyze acyl-CoA, and then the reaction mixture is filteredthrough a Unifilter-96, GF/C plate (PerkinElmer, Waltham, Mass.) using aFilterMate cell harvester (PerkinElmer, Waltham, Mass.). The 96-wellGF/C filter plate is subsequently washed with distilled water to removeexcess [¹⁴C] malonyl-CoA and dried, after which 25 μl of MICROSCINT 0 isadded to each well and radioactivity determined.

7.1.2 Fatty Acid Elongation Assay in Mouse Hepatocytes

Mouse hepatoma 112.35 cells are grown on 24-well plates in Dulbecco'smodified Eagle's medium (DMEM) (Invitrogen, Carlsbad, Calif.)supplemented with 200 nM dexamethasone and 4% heat-inactivated fetalbovine serum (FBS) at 33° C. under 5% CO₂ in a humidified incubator. Thetest compound is dissolved in medium and incubated with subconfluentH2.35 cells for 1 hour at 33° C. [1-¹⁴C]palmitic acid (PerkinElmerJapan, Kanagawa, Japan) is added to each well to a final concentrationof 0.8 μCi/ml to detect elongase activity. After 4 hours of incubationat 33° C., the culture medium is removed, and the labeled cells arewashed with chilled PBS (3×0.5 ml) and dissolved in 250 μl of 2M sodiumhydroxide. The cell lysate is incubated at 70° C. for 1 hour tohydrolyze radiolabeled cellular lipids. After acidification with 100 μlof 5M HCl, fatty acids are extracted with 300 μl of acetonitrile.Radiolabeled palmitic acid (16:0), palmitoleic acid (16:1), stearic acid(18:0), and vaccenic acid:oleic acid (18:1) are quantified byreversed-phase radio-HPLC (RI-HPLC). The identity of the labeled fattyacids is determined by comparing the retention times with known fattyacid standards. Elongation activity was monitored as the elongationindex (EI) which was the ratio of radiolabeled C18 (C18:0+C18:1) to C16(C16:0+±C16:1) estimated from each peak area measured by RI-HPLC.

7.2 In Vivo Assays

7.2.1 [¹⁴C[Palmitate Assay in Mouse Liver

The in vivo efficacy of ELOVL6 inhibitor is determined by following theconversion of radiolabeled 16:0 to 16:1, 18:0, and 18:1 in mice. MaleC57BL/6J mice are orally administrated with ELOVL6 inhibitor and 1 hourlater, the radioactive tracer, [1-¹⁴C] palmitic acid, isinterperitoneally administered at 10 μCi/body. For time-course study ofthe pharmacodynamic effect, [1-¹⁴C]palmitic acid is administered 1, 8 or12 hours after administration of test compounds. At 1 hour post-dosingof the radioactive precursor, animals are anesthetized with isoflurane(4%) and sacrificed for blood collection from the vena cava. Liver (50mg) is harvested and incubated in potassium hydroxide/ethanol (2 ml/1.4ml) at 70° C. for 1 hour. The nonacid-lipid is extracted by 4 ml ofpetroleum ether and discarded. Fatty acids are extracted by 2 ml ofpetroleum ether following saponification by 2 ml of 6 M HCl. The etherphase containing the fatty acid fraction is evaporated under nitrogengas and reconstituted in methanol to measure the radioactivity byRI-HPLC. The radioactivity corresponding to each fatty acid isquantified to calculate the EI.

7.2.2 In Vivo Efficacy in Diet-Induced Obesity (DIO) Alice

Male C57BL/6J mice are maintained on a high-fat diet with ad libitiumaccess to water (D12492, Research Diets, Inc., NJ) for 7 months. Miceare orally administered ELOVL6 inhibitor (dissolved in 0.5%methylcellulose) twice daily (09:30 and 18:30) for 14 days at 30 mg/kgdose. At day 13, body composition is determined and an intraperitonealglucose tolerance test (0.5 kg/g glucose) is performed. At day 14, miceare sacrificed. At 4 hour post-final dosing of ELOVL6 inhibitor, miceare anesthetized and the liver tissues are immediately isolated,weighed, frozen in liquid nitrogen and stored at −80° C. until use.Plasma is prepared and glucose, insulin and leptin are measured usingcommercially available assay kits (Glucose, KyowaMedex, Tokyo, Japan;leptin and insulin, Morinaga, Tokyo, Japan). Liver tissues are isolatedfor the measurement of triglyceride contents and fatty acid composition.For hepatic triglyceride contents, isolated tissues are homogenized in 2ml distilled water, followed by the addition of 6 ml chloroform/methanol(2:1). After centrifugation, the chloroform phase is transferred to anew glass tube containing 1 ml of distilled water and then 3 mlchloroform is added. The lower phase is collected after centrifugationand evaporated to dryness. Extracts are dissolved in 2-propanol and thetriglyceride concentration is measured enzymatically (Determiner TGII,Kyowa Medex, Tokyo Japan). For hepatic fatty acid composition, the liversamples are incubated in 100-fold volume (w/v) of 5 M NaOH/ethanol (1:1)at 60° C.

After 2 hour incubation, 500 μl of 5 MC17:0 (internal standard) areadded to all hydrolysates. The fatty acid compositions are analyzed asfollowing. The fatty acids in the tissue hydrolysate are derivatizedwith 2-nitrophenylhydrazine (2-NPH), and these derivatives are purifiedusing an Oasis HLB column. An aliquot (10 μl) of the eluate is injectedinto the HPLC apparatus for analysis. HPLC analysis is performed with aShimazu 10Avp system (Kyoto, Japan), equipped with a UV detector(SPD-10Avp), two pumps (LC-10ADvp), an auto-sampler (SIL-10ADvp), and acolumn oven (CTO-10ACvp). The mobile phase consist of CH₃CN-water(80:20, flow rate: 0.6 ml/min). The separation is performed with aCAPCELL PAK C18 MGII (2.0 mm i.d.×150 mm, 5 μm) at 35° C. and the UVabsorbance is subsequently measured at 400 nm. The elongation indexrepresents the ratio of C18 (C18:0+C18:1) to C16 (C16:0+C16:1) which isquantified from each fatty acid amount.

7.2.3 In Vivo Efficacy in KKAy Mice

Male KKAy mice given a regular diet (CE2, CLEA Japan) are orallyadministered ELOVL6 inhibitor (dissolved in 0.5% methylcellulose) twicedaily (09:30, 18:30) for 28 days at 30 mg/kg dose. At day 21, anintraperitoneal glucose tolerance test (0.5 kg/g glucose) is performed.At day 28, body composition is determined and mice are sacrificed.Plasma parameters, hepatic triglyceride contents, and fatty acidcomposition are measured as described above.

72.4 Pharmacokinetic Studies in Mice

Single doses of test compound at 10 mg/kg body weight are administeredorally to C57BL/6J mice by gavage in a vehicle of 0.5% methylcelluloseaqueous suspension. Blood samples from the abdominal vein and liversamples are obtained 2 hours after administration. In the case of an indiet regimen, mice are dosed with 100 mg/kg at 17:00 and fed a dietcontaining 0.13% test compound overnight. Then mice are sacrificed thenext morning. Blood samples are centrifuged to separate the plasma.Liver samples are homogenized with phosphate-buffered saline (pH 7.4).Each sample is deproteinized with ethanol containing an internalstandard. Test compound and the internal standard are detected by liquidchromatography mass spectrometry/mass spectrometry (Quattro Ultima massspectrometer, Waters, Milford, Mass.) in positive ionization mode usingan electrospray ionization probe, and their precursor to productioncombinations are monitored using the Multiple Reaction Monitoring mode.

EXEMPLIFICATION

The disclosed compounds can be prepared in a number of ways well knownto one skilled in the art of organic synthesis. More specifically,disclosed compounds can be prepared using the reactions and techniquesdescribed herein. In the description of the synthetic methods describedbelow, it is to be understood that all proposed reaction conditions,including choice of solvent, reaction atmosphere, reaction temperature,duration of the experiment and workup procedures, can be chosen to bethe conditions standard for that reaction, unless otherwise indicated.It is understood by one skilled in the art of organic synthesis that thefunctionality present on various portions of the molecule should becompatible with the reagents and reactions proposed. Substituents notcompatible with the reaction conditions will be apparent to one skilledin the art, and alternate methods are therefore indicated. The startingmaterials for the examples are either commercially available or arereadily prepared by standard methods from known materials.

Synthetic Methods General Synthesis

(i) Synthesis of Tetrazolone Intermediate

Step S-1:

A solution of aniline (A) (1.0 equiv) and triethylamine (1.0 equiv) indry dichloroethane (DCE) (0.2 M) under argon is treated with triphosgene(0.4 equiv) and heated to reflux for 2 hours, or until the reaction isdetermined to be complete by LCMS or TLC. The reaction is then cooled toroom temperature, diluted with dichloromethane, washed with 1N HCl (aq)and brine, dried over MgSO₄, and then filtered and concentrated invacuo. The desired isocyanate (B) is used in the next step withoutpurification.

Step S-2:

A mixture of the isocyanate (B) (1.0 equiv) and trimethylsilyl azide(2.0 equiv) is heated to reflux for 24 hours, or until the reaction isdetermined to be complete by LCMS or TLC. The excess of azide is removedin vacuo and the residue is crystallized from toluene or methanol toprovide the desired tetrazolone intermediate (C) as a white solid.

(ii) Synthesis of the Amine Intermediate

Step S-3:

Copper iodide (1 equiv) and cesium carbonate (2.0 equiv) are added to amicrowave vial, and the vial is evacuated and filled with argon threetimes. An aryl iodide (D-1) in dry dimethylformamide (0.6 M), an alkylamine (R¹¹NH₂) (2.0 equiv) and 2-isobutyrylcyclohexanone (0.2 equiv) arethen added to the vial, the vial is sealed and the resulting mixture isheated to 100° C. under microwave irradiation for 2 hours, or until thereaction is determined complete by LCMS or TLC. At that time, the vialis cooled to room temperature, and the reaction mixture is diluted withethyl acetate and filtered through a pad of Celite with the aid of ethylacetate. The filtrate is washed with brine (3×), dried over MgSO₄,filtered and concentrated in vacuo. The residue is purified by flashchromatography on silica gel (ethyl acetate/hexanes as elutant) toprovide the desired amine intermediate (E).

Step S-4:

To a mixture of nitro compound (D-2) (1.0 equiv) and iron (15.0 equiv)in 1:1 absolute ethanol/dry tetrahydrofuran solution (0.8 mL/mmol ofester) is added water (10 uL/ml of solvent). The mixture is then cooledto 0° C. and a solution of concentrated sulfuric acid (4.0 equiv) inwater (1.2 ml/mmol of ester) is added dropwise to the mixture. Thereaction is warmed to room temperature and stirred for 1 hour, or untilthe reaction is determined complete by LCMS or TLC. The reaction is thenfiltered through a pad of Celite® with the aid of ethyl acetate. Thefiltrate is diluted with brine, saturated aqueous sodium bicarbonatesolution and additional ethyl acetate, the organic and aqueous layersare separated, and the organic layer is washed with saturated sodiumbicarbonate solution, brine, dried over MgSO₄, filtered and concentratedin vacuo. The residue is purified by flash chromatography on silica gel(ethyl acetate/hexanes as elutant) to provide D-3.

Step S-5:

To a stirred solution of D-3 (1.0 equiv) in acetic acid (0.2 M) is addeda carbonyl compound (10.0 equiv of a ketone or aldehyde) and sodiumborohydride (10.0 equiv), and the resulting mixture is stirred at roomtemperature for 1 hour, or until the reaction is determined complete byLCMS or TLC. The reaction is then diluted with ethyl acetate, and washedwith saturated aqueous sodium bicarbonate solution (5×) and brine (2×),and the organic layer is dried over MgSO₄, filtered and concentrated invacuo. The residue is purified by flash chromatography on silica gel(ethyl acetate/hexanes) to provide the desired amine intermediate (E).

(iii) Coupling of Tetrazolone Intermediate and Amine Intermediate

Step S-6:

To a solution of amine intermediate (E) (1.3 equiv) in drytetrahydrofuran (0.5 M) under argon is added triethylamine (1.3 equiv)followed by triphosgene (0.7 equiv). The resulting heterogeneous mixtureis stirred at room temperature for 15 minutes, treated withdimethylaminopyridine (DMAP) (1.0 equiv) and the tetrazoloneintermediate (C) (1.0 equiv), diluted with dry tetrahydrofuran (to 0.25M final concentration with respect to amine) and heated to reflux for 1hour, or until the reaction is determined to be complete by LCMS or TLC.The reaction mixture is cooled to room temperature and diluted withethyl acetate and brine, and the aqueous and organic layers areseparated. The organic layer is washed with brine, 10% aqueous HCl, thenbrine, and dried over MgSO₄, filtered and concentrated in vacuo. Theresidue is purified by flash chromatography on silica gel (ethylacetate/hexanes) to provide the desired tetrazolone product (F).

(iv) Scope of the General Synthetic Method

The general synthetic method is not intended to be limited to thecoupling of amine intermediates such as (E) with phenyl tetrazoloneintermediates such as (C) in the formation of compounds provided herein.For example, other amines and tetrazolones have been coupled using thegeneral methods described above to provide a wide variety of tetrazolonecompounds, e.g., such as the tetrazolone compounds provided in Tables1-4.

Exemplary Syntheses of Compounds

(i) Synthesis of Tetrazolone Compounds 7 and 8

Compound 3:

A mixture of commercially available 2,6-difluorophenyl isocyanate (2)(1.0 equiv) and trimethylsilyl azide (2.0 equiv) was heated to refluxfor 12 hours. The excess of azide was removed in vacuo and the residuewas crystallized from toluene to provide compound (3) as white needles.

Compound 5:

To a solution of commercially available 5-iodo-2-methoxybenzoic acid (4)(1.0 equiv) in dry dimethylformamide (DMF) (0.5 M) under argon was addedsolid potassium carbonate (1.5 equiv) followed by benzyl bromide (1.1equiv), and the resulting mixture was stirred at room temperature for 4hours, acidified with 10% HCl, and diluted with brine and ethyl acetate.The aqueous and organic layers were separated and the organic layer waswashed with brine (5×), dried over MgSO₄, filtered and concentrated invacuo to afford an oil, compound (5), which was used without furtherpurification.

Compound 6:

Copper iodide (1 equiv) and cesium carbonate (2.0 equiv) were added to amicrowave vial and the vial was evacuated and filled with argon threetimes. Compound (5) in dry dimethylformamide (0.6 M), isopropylamine(2.0 equiv) and 2-isobutyrylcyclohexanone (0.2 equiv) were then added tothe vial, the vial was sealed and the resulting mixture was heated to100° C. under microwave irradiation for 2 hours. At that time, the vialwas cooled to room temperature and the reaction mixture was diluted withethyl acetate and filtered through a pad of Celite® with the aid ofethyl acetate. The filtrate was washed with brine (3×), dried overMgSO₄, filtered and concentrated in vacuo. The residue was purified byflash chromatography on silica gel (ethyl acetate/hexanes) to providecompound (6).

Compound 7:

To a solution of compound (6) (1.5 equiv) in dry dichloromethane (0.5 M)under argon was added triethylamine (3.0 equiv) followed by triphosgene(1.5 equiv). The resulting heterogeneous mixture was stirred at roomtemperature for 15 minutes, concentrated in vacuo, and the residue wastaken up in dry toluene (0.25 M with respect to amine) and treated withdimethylaminopyridine (DMAP) (1.0 equiv) and the compound (3) (1.0equiv). The resulting mixture was heated to reflux for 1 hour, cooled toroom temperature and diluted with ethyl acetate and brine, and theaqueous and organic layers were separated. The organic layer was washedwith brine, 10% aqueous HCl, then brine, and dried over MgSO₄, filteredand concentrated in vacuo. The residue was purified by flashchromatography on silica gel (ethyl acetate/hexanes) to provide compound(7) as a white foam.

Compound 8:

To a solution of compound 7 (1.0 equiv) in 2:1 methanol/THF (0.1 M)under argon was added 5% Pd/C (0.1 equiv) and the inert atmosphere wasreplaced with hydrogen. The resulting mixture was stirred at roomtemperature for 30 minutes, filtered through a pad of Celite with theaid of methanol and the resulting filtrate was concentrated in vacuo.The residue was purified by preparative HPLC (0.1% formic acid inacetonitrile/water) to provide compound (8) as a white solid.

(ii) Synthesis of Tetrazolone Compounds 15 and 16

Compound 10:

To a stirred solution of commercially available compound (9) (1.0 equiv)in 1:1 THF:H₂O (0.6 M) under nitrogen was added LiOH (5.0 equiv) at roomtemperature. The reaction was heated to 80° C. for two hours. Theresulting solution was cooled to room temperature and the volume wasreduced by rotary evaporation. The resulting solution was acidified with2N HCl to pH=1, extracted with ethyl acetate, dried with sodium sulfate,and concentrated via rotary evaporation to provide compound (10) whichwas advanced without purification.

Compound 11:

To a stirred solution of (10) (1.0 equiv) in 20:1 acetone:DMF (0.2 M)under nitrogen was added potassium carbonate (1.5 equiv),p-methoxybenzyl chloride (1.0 equiv), and sodium iodide (catalytic) atroom temperature. The resulting mixture was heated to 70° C. for sixhours. The resulting solution was cooled to room temperature and thevolume was reduced by rotary evaporation. The resulting solution wasdiluted with hexanes and ethyl acetate, washed with water, dried withsodium sulfate and concentrated via rotary evaporation. The resultingoil was purified by silica gel column chromatography (hexanes to 25%ethyl acetate/hexanes) to afford compound (11) as an oil.

Compound 12:

To a stirred solution of compound (11) (1.0 equiv) in DMF (0.12M) atroom temperature in a microwave vial was added cesium carbonate (2.00equiv), copper iodide (0.1 equiv), 2-isobutyrylcyclohexanone (0.2equiv), and cyclohexylamine (3.0 equiv). The resulting suspension washeated in the microwave at 100° C. for 1 hr. The resulting suspensionwas cooled to room temperature, diluted with ethyl acetate, washed withdilute aqueous lithium chloride, and washed with water. The organiclayer was dried with sodium sulfate and concentrated via rotaryevaporation to afford a green oil which was purified by silica gelcolumn chromatography (hexanes to 1:2 ethyl acetate/hexanes) to affordcompound (12) as an oil.

Compound 14:

To a stirred solution of commercially available compound (13) (1.00equiv) was added azidotrimethylsilane (4.00 equiv). The resultingsuspension was heated at 90° C. for six hours. The hot reaction solutionwas poured directly into a beaker containing toluene:ice (1:1). Theresulting yellow precipitate was collected via vacuum filtration andwashed with cold toluene to afford compound (14) as a yellow solid.

Compound 15:

To a stirred solution of compound (12) (1.2 equiv) in dichloromethane(0.5M) at 0° C. was added triethylamine (3.0 equiv) and triphosgene (1.0equiv). The ice bath was removed and the solution was stirred at roomtemperature for 15 minutes followed by concentrated by rotaryevaporation. The resulting foam was dissolved in toluene (0.4M) followedby addition of compound (14) (1.0 equiv) and dimethylaminopyridine (1.0equiv). The resulting suspension was heated at 70° C. for sixteen hours.The solution was cooled to room temperature, diluted with ethyl acetate,and washed with water. The organic layer was dried with sodium sulfateand concentrated via rotary evaporation. The resulting oil was purifiedby silica gel column chromatography (ethyl acetate/hexanes) to affordcompound (15) as a white solid.

Compound 16:

To a stirred solution of compound (15) (1.0 equiv) in CH₂Cl₂ (0.1 M)under nitrogen was added trifluoroacetic acid (10.0 equiv) followed byanisole (1.0 equiv) at room temperature. The reaction was run at roomtemperature for ten minutes. The reaction was diluted withdichloromethane and neutralized with aqueous sodium bicarbonatesolution. The organic layer was dried with sodium sulfate andconcentrated via rotary evaporation. The resulting oil was purified bypreparative HPLC (0.1% formic acid in acetonitrile/water) andlyophilized to give compound (16) as a white powder.

(iii) Synthesis of Tetrazolone Compounds 17 and 18

Compound 17:

To a stirred solution of compound (16) (1.0 equiv) in MeOH (0.02 M)under nitrogen was added palladium hydroxide on carbon (0.2 equiv)followed by vacuum evacuation and exposure to hydrogen gas (1 atm,balloon). The reaction was run at room temperature overnight. Theresulting solution was filtered thru celite, washed with dichloromethaneand concentrated via rotary evaporation. The resulting oil was purifiedby preparative HPLC (0.1% formic acid in acetonitrile/water) andlyophilized to afford compound (17) as a white solid.

Compound 18:

To a stirred solution of compound (17) was added formic acid (500equiv). The reaction was heated at 100° C. for 1 hour. After cooling toroom temperature, the reaction was diluted with ethyl acetate and theorganic layer was washed with water, dried with sodium sulfate, andconcentrated via rotary evaporation. The resulting oil was purified bypreparative HPLC (0.1% formic acid in acetonitrile/water) andlyophilized to afford compound (18) as a white solid. Compound (18) isdepicted above as one compound, but it exists in equilibrium as amixture of tautomers as shown below.

(iv) Synthesis of Tetrazolone Compounds 24 and 25

Compound 20:

To a solution of commercially available 4-oxocyclohexanecarboxylic acid(19) (1.0 equiv) in dry DMF (0.5 M) under argon was added solidpotassium carbonate (1.5 equiv) followed by benzyl bromide (1.1 equiv).The resulting mixture was stirred at room temperature for 4 hours,acidified with 10% HCl, diluted with brine and ethyl acetate, and thelayers were separated. The organic layer was washed with brine (5×),dried (MgSO₄), filtered and concentrated in vacuo to afford a solidwhich was used without further purification.

Compound 21:

A solution of benzyl 4-oxocyclohexanecarboxylate (20) (1.0 equiv) and a2M ethylamine solution in THF (2.0 equiv) in dry DCE (1.4 M) under argonwas treated with sodium triacetoxyborohydride (1.5 equiv) and theresulting mixture was stirred at rt for 1 h, and quenched with saturatedaqueous sodium bicarbonate solution. The layers were separated and theaqueous one extracted with DCM (3×). The combined organic layers werewashed with brine, dried (MgSO₄), filtered and concentrated in vacuo.The residue was purified by flash chromatography on silica gel (ethylacetate/hexanes) to provide compound (21) as an oil.

Compound 23:

A mixture of commercially available 2,6-dichlorophenyl isocyanate (22)(1.0 equiv) and trimethylsilyl azide (2.0 equiv) was heated to refluxfor 12 hours. The excess of azide was removed in vacuo and the residuewas crystallized from methanol to provide compound (23) as whiteneedles.

Compound 24:

To a solution of compound (21) (1.3 equiv) in dry tetrahydrofuran (0.5M) under argon was added triethylamine (1.3 equiv) followed bytriphosgene (1.3 equiv). The resulting heterogeneous mixture was stirredat room temperature for 15 minutes diluted with dry tetrahydrofuran(0.25 M with respect to amine) and treated with DMAP (1.0 equiv) andcompound (23) (1.0 equiv). The resulting mixture was heated to refluxfor 1 hour, cooled to room temperature and diluted with ethyl acetateand brine, and the aqueous and organic layers were separated. Theorganic layer was washed with brine, 10% aqueous HCl, then brine, anddried over MgSO₄, filtered and concentrated in vacuo. The residue waspurified by flash chromatography on silica gel (ethyl acetate/hexanes)to provide compound (24) as a white solid.

Compound 25:

To a solution of compound (24) (1.0 equiv) in methanol (0.1 M) underargon was added 10% Pd(OH)₂ on carbon (0.1 equiv) and the inertatmosphere was replaced with hydrogen. The resulting mixture was stirredat room temperature for 2 h, filtered through a pad of Celite with theaid of methanol, and the resulting filtrate concentrated in vacuo. Theresidue was purified by preparative HPLC (0.1% formic acid inacetonitrile/water) to provide compound (25) as a white solid.

(v) Synthesis of Tetrazolone Compounds 27 and 28

Compound 27:

Commercially available tert-butyl piperidin-3-ylcarbamate (26) (1.3equiv) in dry tetrahydrofuran (0.5 M) under argon was addedtriethylamine (1.3 equiv) followed by triphosgene (1.3 equiv). Theresulting heterogeneous mixture was stirred at room temperature for 15minutes, diluted with dry tetrahydrofuran (0.25 M with respect to amine)and treated with DMAP (1.0 equiv) and compound (23) (1.0 equiv). Theresulting mixture was heated to reflux for 1 hour, cooled to roomtemperature and diluted with ethyl acetate and brine, and the aqueousand organic layers are separated. The organic layer was washed withbrine, 10% aqueous HCl, then brine, and dried over MgSO₄, filtered andconcentrated in vacuo. The residue was used without furtherpurification. An analytical sample was obtained by preparative HPLC(0.1% formic acid in acetonitrile/water) to provide compound (27) as awhite solid.

Compound 28:

Compound (27) (1.0 equiv) was dissolved in dry dichloromethane (0.2 M)at 0° C. and was treated dropwise with TFA (3:1 DCM/TFA). The resultingsolution was stirred at room temperature for 1 h and was concentrated invacuo. The amine salt was then taken up in dry dichloromethane, cooledto 0° C. and treated with excess of acetyl chloride (10.0 equiv) andtriethylamine (1.0 equiv). The resulting mixture was stirred at 0° C.for 15 min, quenched with 10% HCl and extracted with DCM (2×). Thecombined organic extracts were washed with brine, dried (MgSO₄),filtered and concentrated in vacuo. The residue was purified bypreparative HPLC (0.1% formic acid in acetonitrile/water) to providecompound (28) as a white solid.

(vi) Synthesis of Tetrazolone Compounds 32 and 33

Compound 30:

To a stirred solution of 5-amino-2-pyridinecarboxylic acid (29) (1.0equiv) in AcOH (0.9 M) under nitrogen was added cyclohexanone (1.1equiv) and sodium triacetoxyborohydride (1.1 equiv) at room temperature.The resulting mixture was stirred at room temperature for 72 hours.Methanol was added and the resulting solution was concentrated by rotaryevaporation. The resulting oil was dissolved in CHCl₃:IPA (4:1) andwater, and the organic layer was concentrated in vacuo to providecompound (30) which was advanced without purification.

Compound 31:

To a stirred solution of compound (30) (1.0 equiv) in 2:1 CH₂Cl₂:DMSO(0.13M) was added AgO (3.0 equiv) followed by benzyl bromide (1.1equiv). The reaction was run at room temperature overnight. Theresulting solution was filtered through a celite pad, diluted withCH₂Cl₂, washed with water, dried with sodium sulfate, and concentratedby rotary evaporation. The resulting oil was purified by silica gelchromatography (hexanes to 50% ethyl acetate/hexanes) to afford compound(31) as an off-white foam.

Compound 32:

To a stirred solution of compound (31) (0.9 equiv) in dichloromethane(0.5M) at 0 C was added triethylamine (3.0 equiv) and triphosgene (1.0equiv). The ice bath was removed and the solution was stirred at roomtemperature for 15 minutes followed by concentrated by rotaryevaporation. The resulting foam was dissolved in toluene (0.4M) followedby addition of compound (3) (1.0 equiv) and dimethylaminopyridine (1.0equiv). The resulting suspension was heated at 90° C. for two hours. Thesolution was cooled to room temperature, diluted with ethyl acetate, andwashed with water. The organic layer was dried with sodium sulfate andconcentrated via rotary evaporation. The resulting oil was purified bysilica gel column chromatography (hexanes to 50% ethyl acetate/hexanes)to afford compound (32).

Compound 33:

To a stirred solution of (32) (1.00 equiv) in methanol (0.1M) at roomtemperature was added 20% palladium hydroxide on carbon (˜0.2 equiv).The reaction was evacuated and flushed with nitrogen followed bycharging with hydrogen gas. After stirring for one hour, the reactionwas filtered through celite, washed with dichloromethane, andconcentrated by rotary evaporation. The resulting oil was purified bysilica gel column chromatography (dichloromethane to 9:1dichloromethane:methanol) to afford 33 as a clear oil.

(vii) Synthesis of Tetrazolone Compounds 40 and 41

Compound 36:

To a stirred suspension of commercially available compound (34) (1.0equiv) and potassium carbonate (1.5 equiv) in dry DMF (0.3 M) undernitrogen was added benzyl bromide (1.2 equiv) at room temperature. Thereaction was stirred for 19 hours, additional potassium carbonate (1.5equiv) was added followed by phenol (1.2 equiv) and stirring wascontinued for 24 h. The resulting suspension was filtered through a padof Celite with aid of AcOEt, the filtrate was diluted with additionalAcOEt and was washed with brine and 10% HCl. The aqueous layer wasback-extracted with AcOEt and the combined organic layers were washedwith brine (3×), dried (MgSO₄), filtered and concentrated in vacuo. Anorange oil was obtained, which was purified by flash chromatography(Hexane/AcOEt 9:1) to render compound 36 as a thick yellow oil (90%yield).

Compound 37:

To a stirred solution of (36) (1.0 equiv) and iron powder (15.3 equiv)in 1:6 absolute EtOH/THF (0.3 M) cooled to 0° C., was dropwise added asolution of H₂SO₄ (0.6 ml/mmol) in H₂O (1.8 ml/mmol). The resultingmixture was stirred at rt for 1 h, filtered through a pad of Celite withthe aid of AcOEt, and the filtrate diluted with brine and additionalAcOEt. The layers were separated, the organic one was washed with brine,saturated sodium bicarbonate solution, brine, dried (MgSO₄), filteredand concentrated in vacuum. Compound (37) was obtained as a pale yellowoil which crystallized upon standing.

Compound 38:

To an stirred suspension of aniline (37) in glacial AcOH (0.3M) wasadded acetone (5 equiv) followed by sodium borohydride (2 equiv). Theresulting mixture was stirred at rt for 1 h, diluted with AcOEt andwashed with brine (3×), saturated sodium bicarbonate solution (6×, untilbasic pH) and brine (2×). The organic layer was dried (MgSO₄), filteredand concentrated in vacuo. Compound (38) was obtained as an oil afterpurification by flash chromatography (Hexane/AcOEt 91 to 3:1) (80% yieldover 2 steps).

Compound 40:

To a stirred solution of compound (38) (1.0 equiv) in dichloromethane(0.3 M) at 0° C. was added triethylamine (2.0 equiv) and triphosgene(0.7 equiv). The ice bath was removed and the solution was stirred atroom temperature for 15 minutes and was concentrated in vacuo. Theresulting foam was dissolved in toluene (0.3 M) followed by addition ofcompound (39) (1.1 equiv) and dimethylaminopyridine (1.0 equiv). Theresulting suspension was heated to reflux for 3 h, was then cooled toroom temperature, diluted with ethyl acetate, and washed with water, 10%hydrochloric acid solution and brine. The organic layer was dried(MgSO₄), filtered and concentrated in vacuo. The resulting crude waspurified by flash chromatography (Hexane/AcOEt 5:1 to 2:1) to affordcompound (40) as a pale yellow foam (66% yield).

Compound 41:

To a solution of compound (40) (1.0 equiv) in 1:1 methanol/AcOEt (0.3 M)under argon was added 10% Pd/C (0.15 equiv) and the inert atmosphere wasreplaced with hydrogen. The resulting mixture was stirred at roomtemperature for 30 minutes, filtered through a pad of Celite with theaid of methanol and the resulting filtrate was concentrated in vacuo.The residue was purified by flash chromatography (Hexane/AcOEt 2:1 to1:4 and DCM/MeOH 9:1) to provide compound (41) as a white foam (58%yield) [M+1]⁺=495.1.

(viii) Synthesis of Tetarzolone Compounds 51 and 52

Compound 44:

Compound (44) was prepared in 64% yield via methylation of commerciallyavailable 4-flurosalicilic acid (42) followed by nitration as describedin the literature (Del Corona, L.; Signorelli, G.; Pinzetta, A.; Coppi,G. Eur. J. Med. Chem. 1993, 28: 419-425).

Compound 45:

To a solution of compound (44) (1.0 equiv) in dry DMF (0.9 M) was addedphenol (1.1 equiv) and potassium carbonate (1.5 equiv) and the resultingmixture was stirred at rt for 1 h, the solid filtered and washed withAcOEt, the filtrate further diluted with AcOEt and washed with 10% HCland brine (5×). The aqueous layer was back-extracted with AcOEt, whichwas washed with brine. The combined organic layers were dried (MgSO₄),filtered and concentrated in vacuo. Compound (45) was obtained as anoil, which was used directly.

Compound 46:

A solution of methyl ester (45) (1.0 equiv) in 2:2:5 MeOH/THF/H₂O (0.3M) was added LiOH (1.5 equiv). The suspension was stirred at 60° C. for1.5 h. The reaction mixture was allowed to cool to rt, diluted withbrine and extracted with diethyl ether. The aqueous layer was acidifiedwith 10% HCl and extracted with AcOEt (3×). The combined AcOEt layerswere washed with brine, dried (MgSO₄), filtered and concentrated invacuo. Compound 46 was obtained as a pale yellow foam, which was useddirectly.

Compound 47:

To a stirred solution of acid (46) (1.0 equiv) and potassium carbonate(1.5 equiv) in dry DMF (0.6 M) under nitrogen was added benzyl bromide(1.1 equiv) at room temperature. The reaction was stirred for 19 hours,filtered through a pad of Celite with aid of AcOEt, the filtrate wasacidified with 10% HCl, diluted with brine and extracted with AcOEt(3×). The combined organic extracts were washed with brine, dried(MgSO₄), filtered and concentrated in vacuo. The residue (oil) was useddirectly.

Compound 48:

To a stirred solution of (47) (1.0 equiv) in 2:1 THF/AcOH (0.6 M) cooledto 0° C., was portionwise added zinc powder (10.0 equiv). The resultingmixture was stirred at 0° C. for 1 h, filtered through a pad of Celitewith the aid of AcOEt and the filtrate was diluted with brine andadditional AcOEt. The layers were separated, the organic one was washedwith brine, saturated sodium bicarbonate solution, brine, dried (MgSO₄),filtered and concentrated in vacuo. Compound (48) was obtained as a paleyellow oil which crystallized upon standing.

Compound 49:

To an stirred suspension of aniline (48) in glacial AcOH (0.3 M) wasadded cyclohexanone (4.0 equiv) followed by sodium borohydride (2.5equiv). The resulting mixture was stirred at rt for 1 h, diluted withAcOEt and washed with brine (3×), saturated sodium bicarbonate solution(6×, until basic pH) and brine (2×). The organic layer was dried(MgSO₄), filtered and concentrated in vacuo. The residue was purified byflash chromatography (Hexane/AcOEt 9:1) to give compound (49) as an oil(46% yield over 5 steps).

Compound 51:

To a stirred solution of compound (49) (1.0 equiv) in dichloromethane(0.2 M) at 0° C. was added triethylamine (2.0 equiv) and triphosgene(0.7 equiv). The ice bath was removed and the solution was stirred atroom temperature for 15 minutes and was concentrated in vacuo. Theresulting foam was dissolved in toluene (0.2 M) followed by addition ofcompound (50) (1.1 equiv) and dimethylaminopyridine (1.0 equiv). Theresulting suspension was heated to reflux for 2 h, was then cooled toroom temperature, diluted with ethyl acetate, and washed with water, 10%hydrochloric acid solution and brine. The organic layer was dried(MgSO₄), filtered and concentrated in vacuo. The resulting product waspurified by flash chromatography (Hexane/AcOEt 9:1 to 2:1) to affordcompound (51) as a crystalline solid (22% yield).

Compound 52:

To a solution of compound (51) (1.0 equiv) in 1:1 methanol/THF (0.1 M)under argon was added 10% Pd/C (0.15 equiv) and the inert atmosphere wasreplaced with hydrogen. The resulting mixture was stirred at roomtemperature for 1 h, filtered through a pad of Celite with the aid ofmethanol and the resulting filtrate was concentrated in vacuo. Compound(52) was obtained as a crystalline solid (99% yield).

Biological Assays Preparation of Human FASN Protein

Human FASN protein (SEQ ID NO. 1) was purified from SKBR3 cells usingprocedures modified from those in Jayakumar et al., PNAS 1995,92:8695-8699. SKBR3 cells were obtained from ATCC and grown in DMEM highglucose medium supplemented with 10% FBS, 1 μg/mL bovine pancreasinsulin, 100 U/mL penicillin and 100 μg/mL streptomycin. The confluentcells were trypsinized and washed three times with PBS buffer beforefrozen in liquid N₂ and stored at −80° C. Frozen cells were thawed onice and resuspended in lysis buffer (25 mM Tris-HCl, pH 7.0, 15 mM NaCl,1 mM EDTA, and 1 mM DTT) with protease inhibitors. The cells were lysedby sonication, and the cell debris was removed by centrifugation at20,000 rpm for 30 min. To the supernatant, neutralized saturatedammonium sulfate solution was added to a final concentration of 35%. Thesolution was left on ice for 1 hr, and the precipitated proteins wereharvested by centrifugation at 20,000 rpm for 30 min. The proteins wereredissolved in lysis buffer without NaCl and loaded on a mono Q column.Bound proteins were eluted with a linear gradient of NaCl in lysisbuffer. Each fraction was analyzed by SDS-PAGE and FASN NADPHconsumption assay. The fractions containing FASN were pooled andconcentrated to 2-3 mg/mL. Glycerol was added to 20%, and the proteinwas frozen in liquid N₂ and stored at −80° C.

FASN NADPH Consumption Assay

All chemicals were purchased from Sigma (St. Louis, Mo.). The proceduresof NADPH consumption assay were similar to those described in Cox etal., PNAS 1983, 80:4233-4237. On a 96-well polypropylene microplate,dilution series (typical concentrations 60 nM-1.0 mM) of test compoundswere prepared in DMSO, of which 4.0 μL each was transferred to a blackpolystyrene assay microplate and mixed with 36 μL FASN assay buffer (50mM potassium phosphate, pH 7.0, 1.0 mM EDTA, 0.01% NP-40) plus 5.0 mMfresh DTT. FASN protein (40 μL 150 nM FASN) was added per well, and themicroplate was incubated at 37° C. for 30 min. Enzyme activitymeasurement was initiated by addition of 20 μL 5×substrate mixture tofinal concentrations of 60 nM FASN, 2.4 nM-40 M compound, 0.2 mM NADPH,50 M butyryl-CoA, 0.5 mM malonyl-CoA in 100 μL assay buffer plus 5.0 mMDTT and 4.0% DMSO. NADPH consumption was monitored kinetically byfluorescence (λ_(Ex)=340 nm, λ_(Em)=460 nm) on an EnVision 2100multilabel plate reader (Perkin Elmer, Waltham, Mass.). FASN enzymeactivity (slope) in the presence of 4% DMSO was used as maximum control,whereas background (minimum control) was measured by omission ofmalonyl-CoA in the substrate mixture. Inhibition curves were fitted by alogistic function to yield IC₅₀ values:

$\begin{matrix}{{\% \mspace{14mu} {Inhibition}} = {\left\lbrack {1 - \frac{\left( {{Slope} - {Min}} \right)}{\left( {{Max} - {Min}} \right)}} \right\rbrack \times 100\%}} \\{{\% \mspace{14mu} {Inhibition}} = \frac{100}{1 + \left( {{IC}_{50}/\lbrack I\rbrack_{total}} \right)^{{Hill}\mspace{14mu} {coefficient}}}}\end{matrix}$

Compounds provided herein were found to inhibit FASN activity using thisassay.

Activities obtained by the above-described FASN NADPH Consumption Assayare designated in Tables 1, 2, 3 and 4 with a star (*), wherein “A*”refers to compounds having an IC₅₀ of less than 60 nM; “B*” refers tocompounds having an IC₅₀ of 60 nM to 250 nM, inclusive; “C*” refers tocompounds having an IC₅₀ of greater than 250 nM to 1000 nM, inclusive;“D*” refers to compounds having an IC₅₀ of greater than 1000 nM to10,000 nM, inclusive; and “E*” refers to compounds having an IC₅₀ ofgreater than 10,000 nM.

FASN Scintillation Proximity Flashplate Assay

Acetyl-coenzyme A, malonyl-coenzyme A, NADPH, bovine gamma globulin, andORLISTAT were purchased from Sigma (St. Louis, Mo.).Tris(2-carboxyethyl) phosphine hydrochloride (TCEP) was purchased fromPierce Biotechnologies (Rockford, Ill.). [³H]-acetyl-coenzyme A waspurchased from Moravek Biochemicals (Brea, Calif.). FlashPlate® PLUSphospholipid 96-well scintillant coated microplates were purchased fromPerkin Elmer Life and Analytical Sciences (Shelton, Conn.). The methodof the FASN Scintillation Proximity FlashPlate assay is similar to thatdescribed in Weiss and Glickman Assay Drug Dev Technol 2003, 1:161-6. Ina 96-well polypropylene microplate, a dilution series (typicalconcentrations 60 nM-1.0 mM) of test compounds were prepared in DMSOfollowed by a 20-fold dilution into FASN assay buffer (50 mM potassiumphosphate, pH 7.0, 1.0 mM EDTA, 0.01% NP-40), of which 5.0 L each wastransferred to a FlashPlate® PLUS 96-well plate and mixed with 35 μLFASN assay buffer plus 0.5 mg/mL bovine gamma globulin and 1 mM TCEP.FASN protein (10 μL, 10 nM) was added per well, and the microplate wasincubated at 37° C. for 30 min. 10 μL of 20 mM NADPH was added, and thereaction was initiated by addition of 40 μL substrate mixture to finalconcentrations of 1 nM FASN, 100 M acetyl-coenzyme A, 6 μCi[³H]-acetyl-coenzyme A, 300 M malonyl-coenzyme A, 2 mM NADPH, 0.5 mg/mLbovine gamma globulin, and 1 mM TCEP in a volume of 100 μL per well.Assay plates were incubated for 2 hr at 37° C. and the reaction wasstopped with 2 μL of 2.5 mM stock solution of ORLISTAT in DMSO to ˜50μM. The plates were read in a Wallac 1450 Microbeta Plus liquidscintillation counter (Perkin Elmer, Waltham, Mass.), and counts perminute (CPM) were collected over 2 min. Each inhibitor well CPM wascompared to the maximum FASN enzyme activity (Max) CPM and thebackground (Min) CPM, as measured by omission of FASN enzyme in thebackground well. % Inhibition values were calculated, and curves werefitted by a four-parameter logistic function to yield IC₅₀ values:

${\% \mspace{14mu} {Inhibition}} = {\left\lbrack {1 - \frac{\left( {{Inhibitor} - {Min}} \right)}{\left( {{Max} - {Min}} \right)}} \right\rbrack \times 100\%}$

Compounds provided herein were found to inhibit FASN activity using thisassay.

Activities obtained by the above-described FASN Scintillation ProximityFlashplate Assay are provided in Tables 1, 2, 3 and 4 wherein “A” refersto compounds having an IC₅₀ of less than 15 nM; “B” refers to compoundshaving an IC₅₀ of 15 nM to 100 nM, inclusive; “C” refers to compoundshaving an IC₅₀ of greater than 100 nM to 200 nM, inclusive; “D” refersto compounds having an IC₅₀ of greater than 200 nM to 5000 nM,inclusive; and “E” refers to compounds having an IC₅₀ of greater than5000 nM.

FASN Cellular Flashplate Assay

Compound was added to HCT116 cells 24 hours post-plating (1×10̂6 cellsper well, 6-well plates) and incubated for 24 hours (37 C, 5% CO2). Cellpellets were harvested and washed with PBS. 30 uL FAS buffer minus BGG(50 mM KPB, pH 7.0, 1.0 mM EDTA, 0.01% NP-40, 1 mM TCEP) was added tothe cell pellet, and the cells were lysed by freeze/thaw (3×, liquidnitrogen/42 C water bath) before pelleting debris (20,000 rcf, 15minutes, 4 C). Total protein concentration of the lysates weredetermined (Pierce BCA Total Protein assay, BSA standard) and sampleswere normalized to 1 mg/mL total protein in assay buffer.

Assay setup: 50 uL assay buffer was added to each well of the FlashPlate(PerkinElmer, FlashPlate Plus Phospholipid 96-well scintillant coatedmicroplate). To the wells 10 uL of each of the following were added; 1mM Acetyl-CoA, 3 mM Malonyl-CoA, 0.05 mCi/mL [³H]Acetyl-CoA and 20 mMNADPH for final concentrations of 100 uM Acetyl-CoA, 300 uM Malonyl-CoA,0.5 uCi/well [³H]Acetyl-CoA and 2000 uM NADPH. Finally, 10 uL of FASenzyme was added to the wells either in the form of HCT116 cell lysateor purified FAS for a standard curve. The FlashPlate was incubated at37° C. for 120 min then read on the MicroBeta instrument.

Compounds provided herein were found to inhibit FASN activity using thisassay.

HCV-Replicon Luciferase Assay

DMEM complete medium (Life Technologies) was supplemented with 10% FCS,2 mM glutamin, penicillin and streptomycin, and 1× nonessential aminoacids and pre-warmed in a 37° C. thermostat water bath for use as agrowth medium.

A dish containing HCV-replicon reporter cells, which were kept in a 37°C. CO₂ incubator, was removed from the incubator. The medium wasaspirated, and cells were rinsed with 1 ml PBS. The solution wasdiscarded, and 1 ml of 1.25% trypsin/0.02% EDTA was added toadditionally rinse the cells. The trypsin/EDTA solution was removed witha vacuum pump, and the cells were incubated at 37° C. for 3 to 5minutes. Cell morphology was examined under an inverted microscope untila single cell suspension was clearly visible, then the cell wassuspended with 3 ml of complete medium by gentle pipetting.

Upon suspension, cell numbers were counted with a hematometer, and celldensity was adjusted to 100K/ml by adding appropriate volume of thecomplete medium. One hundred microliters (100 μl) of cell suspension wasadded to each well of a 96-well white plate to provided cell density ofeach of the wells to be 10K/well. The 96-well assay plate was placed ina 37° C. 5% CO₂ incubator for 24 hours.

At the end of the incubation, the plate was removed and test compoundwas added at various desired concentrations using a serial dilution. Theplate was placed back into the 37° C. CO₂ incubator for 48 hours. Afterthe incubation, 30 μl of Stead-Glo Luciferase System® (Promega) reagentwas added to each well and mixed by gentle shaking for 5 minutes toallow thorough cell lysis. Luminescence was measured with Envision®(Perkin-Elmer) with an integration time of 2 seconds.

The tests were conducted using the following compounds:

From this test, it was found that IC₅₀ values for HCV-replicon of thetested compounds range from about 4 nM to about 7.3 μM. These resultsindicate that compounds provided herein are effective inhibitors of HCV.

MTT Assay

DMEM complete medium (Life Technologies) was supplemented with 10% FCS,2 mM glutamin, penicillin and streptomycin, and 1× nonessential aminoacids and pre-warmed in a 37° C. thermostat water bath for use as agrowth medium.

A dish containing HCV-replicon cells, which were kept in a 37° C. CO₂incubator, was removed from the incubator. The medium was aspirated, andthe cells were rinsed with 1 ml PBS. The solution was discarded, and 1ml of 1.25% trypsin/0.02% EDTA was added to additionally rinse thecells. The trypsin/EDTA solution was removed with a vacuum pump, and thecells were incubated at 37° C. for 3 to 5 minutes. Cell morphology wasexamined under an inverted microscope until single cell suspension wasclearly visible, then the cell was suspended with 3 ml of completemedium by gentle pipetting.

Upon suspension, cell numbers were counted with a hematometer, and celldensity was adjusted to 100K/ml by adding appropriate volume of thecomplete medium. One hundred microliters (100 μl) of cell suspension wasadded to each well of a 96-well white plate to provided cell density ofeach of the wells to be 10K/well. The 96-well assay plate was placed ina 37° C. 5% CO₂ incubator for 24 hours.

At the end of the incubation, the plate was removed and test compoundwas added at various desired concentrations using a serial dilution. Theplate was placed back into the 37° C. CO₂ incubator for 48 hours. Afterthe incubation, 10 μl of 5 mg/ml3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) wasadded to each well, and the mixture was incubated in the 37° C. CO₂incubator for 4 hours. One hundred microliters (100 μl) of testingsolution (10% SDS+5% isobutyl alcohol+10 mmol/l HCl) was added directlyto each well, and the mixture was incubated in the 37° C. 5% CO₂incubator overnight. Absorbance at 580/680 nm was measured on SpectraMaxPlus 384® (MDC).

C¹³ Palmitate Assay

HCT-116 tumor cells (ATCC) were labeled for 24 hours in Dulbecco'sminimum Eagle's medium supplemented with 0.5 g/liter [U-¹³C] glucose(Cambridge Isotope Laboratories) and 2.0 g/liter unlabeled glucose(Sigma). Cells were concurrently treated with test compounds at 0.2%DMSO. After 24 hours, cells were harvested with trypsin/EDTA, counted byhemacytometer, washed with PBS, and centrifuged at 2000 rpm for 5minutes. Pellets of 5M cells were stored at −80° C.

The cell pellet was saponified in strong base overnight at 70° C. Themedia was acidified and the palmitic acid extracted with hexanes. Afterdrying the hexane layer, the palimitic acid was reconstituted inmethanol that had been made slightly acidic. The palmitic acid was thenmonitored for C¹³ incorporation via GCMS. Palmitic acid synthesis wasdeemed to have been halted if no C¹³ was incorporated into thepalmitate. Unhindered palmitate synthesis was determined by the amountof C¹³ incorporated in absence of test compound.

Compounds provided herein were found to inhibit palmitate synthesisusing this assay.

Pharmacokinetic Studies Distribution Upon Oral Administration

Prior to the beginning of the tests, a study was conducted to determineif perfusion of the livers is necessary for an accurate quantificationof levels of test compounds in the liver. If necessary, animals wereperfused prior to liver collection. Additionally, a single dose 24-hourtolerability study was conducted on a few animals to verify that a givenlevel of test compound is tolerated.

To the animals, single oral (PO) dose was administered at a volume of 10ml/kg based on most recent body weight. Twenty milligrams per milliliter(20 mg/ml) of test compound in 0.5% Carboxymethylcellulose/5% DMSO/0.5%Tween was used. Vehicle control was also included.

At 0, 15, 30, 60, 120, 240, 360, 480 & 1440 minutes after doseadministration, 3 animals from each group were anesthetized by carbondioxide inhalation and euthanized by cardiac puncture. Blood wascollected into lithium heparin vacutainers, placed on wet ice andcentrifuged at 4° C. within 15 minutes of collection at 10,200 rpm for 5minutes. Plasma was collected into a fresh eppendorf tube, frozen on dryice and stored at −80° C. until PK analysis. Livers were harvested andbisected. Half of each tumor was snap-frozen in liquid nitrogen inpre-weighed Genogrinder tubes, placed on dry ice and stored at −80° C.until pharmacokinetic analysis, and the other half was snap-frozen inliquid nitrogen in pre-weighed Genogrinder tubes, placed on dry ice, andstored at −80° C. until PD analysis.

Inhibition of Human Fasn Scintillation Proximity Flashplate Assay andNADPH Consumption Assay

Compounds prepared following the above described synthetic methods areprovided in Tables 1-4 below. Substituent abbreviations used in Tables1-4 are given in Table 5. Compounds were assayed as inhibitors of humanFASN using the above-described FASN Scintillation Proximity FlashplateAssay (IC₅₀; nM) or FASN NADPH Consumption Assay (*) (IC₅₀; nM). Thecorresponding activity of the isolated compound is also provided alongwith the measured mass (ES⁺).

TABLE 1 STRUCTURE ACTIVITY (M + H)⁺

E* 282.1

B* 384.1

C* 430.1

B* 384.1

A 560.2

C 463.2

B* 420.1

A 446.1

B* 450.2

B* 404.1

C* 414.1

D* 560.2

B* 384.1

A 518.2

B* 414.1

D* 608.2

C* 518.2

B 474.2

B* 504.2

B* 414.1

A 504.2

D 428.2

A 504.2

C 370.1

B* 414.1

C* 414.1

A* 432.1

C* 442.2

C* 470.2

A* 418.1

C* 504.2

A 510.0

D* 442.2

A 456.2

C* 330.1

C* 358.1

B* 408.0

B* 356.1

C* 348.1

C* 384.1

B* 348.1

B* 344.1

C* 374.0

C* 314.1

B* 409.1

A* 424.0

D* 362.1

A 434.1

B* 360.1

A 382.1

B* 362.1

B 448.1

A 524.2

B* 394.1

B* 380.1

A 450.1

B* 410.0

B* 398.1

A 379.1

B* 350.1

B* 410.0

A* 382.1

A 421.1

A 474.2

B* 360.1

A* 358.1

A* 382.1

B* 360.1

C* 357.1

B* 480.1

A 513.2

B* 368.1

A 510.2

B* 360.1

A* 346.1

B* 396.1

B* 391.1

B* 332.1

B* 444.2

A* 494.2

B* 510.2

A 382.1

A* 390.1

B* 524.2

A* 449.2

B* 464.1

B* 510.2

A* 535.1

B* 410.0

B* 360.1

B* 350.1

A 718.3

B* 400.1

B* 444.2

B* 414.0

B* 350.1

A 462.2

B* 468.2

B* 430.1

A 364.1

D* 422.1

C* 412.2

B* 432.1

A 457.1

A 600.2

A* 360.1

B* 433.1

C* 362.1

A 482.1

A 438.1

B* 484.2

B 420.1

A* 391.1

B* 442.0

A 420.1

A* 366.0

A 400.2

A 599.2

A 600.2

A 596.2

A 686.2

A 573.2

A 606.2

A 559.2

A 487.2

A 570.2

A 537.2

B 556.2

A 488.2

A 518.2

A 501.2

A 473.2

A 628.3

A 718.3

B 529.2

A 504.2

A 487.2

A 490.1

A 486.2

A 639.2

B 504.2

A 548.2

A 594.2

A 360.1

A 638.2

A 459.2

A 559.2

A 391.1

A 454.0

A 474.2

A 532.0

A 474.2

A 622.1

A 460.1

A 376.1

B 361.1

A 466.1

A 476.1

A 460.1

A 566.2

A 467.1

A 379.0

A 391.1

A 543.1

B 432.1

A 469.1

A 395.0

A 522.2

A 443.1

A 474.2

A 391.1

A 485.1

B 376.1

A 533.2

B 421.1

B 408.1

D* 452.0

A 456  

B 436.1

A 576.1

B* 456.0

B* 525.0

B* 546.0

B* 576.1

B* 396.0

A* 456.0

C* 349.0

B* 403.0

B* 452.0

B* 546.0

B* 561.0

A 456.0

B* 393.1

B* 423.0

B* 423.0

B 422.0

D* 529.0

B* 542.1

B* 522.1

C* 589.1

D* 499.1

B* 452.0

C* 441.0

A* 561.0

C* 408.1

B* 393.1

C* 378.0

D* 390.0

B* 576.1

B* 364.0

E* 588.1

A 422.0

B* 525.0

C* 444.1

C* 534.2

B 450.2

A 430.1

A 472.2

B 432.1

A 562.2

B 412.2

A 478.2

B 432.1

A 656.2

D 404.2

A 486.2

B 416.1

A* 522.2

B 536.2

A 446.1

D 444.2

A 398.2

B 536.2

B 460.2

D 598.2

A 494.1

B* 414.1

D 430.1

B 508.2

A 536.2

B 446.1

A 504.2

C* 430.2

A 522.2

B 414.2

A 474.2

B 534.2

D 520.2

A 358.1

D* 414.2

A 520.2

A 487.2

A 486.2

B 574.2

A 530.2

A 560.2

A 514.2

B 514.2

B 604.3

B 550.2

D* 464.1

B* 386.1

D 533.2

A 619.2

D 529.2

A 617.2

B 527.2

C 531.2

A 461.2

B 553.2

A 563.2

A 653.2

A 539.2

B 549.2

D 487.2

A 577.2

B* 428.1

B* 442.2

B* 432.2

B* 446.2

B 541.2

B* 406.0

B* 424.0

A 616.2

B 496.1

B 468.2

C 478.2

D* 331.1

C* 333.1

A 445.1

D* 344.1

E* 424.1

C 396.1

E* 358.1

A 496.1

A 508.2

A 536.2

A 522.2

A 566.2

A 552.2

A  448.12

TABLE 2 STRUCTURE ACTIVITY (M + H)⁺

D* 443.1

E* 443.1

E* 328.0

E* 385.1

E* 385.1

E* 399.1

E* 385.1

D* 442.1

E* 439.1

D* 457.1

D* 441.1

D* 385.1

E* 399.1

D* 453.1

E* 386.0

D* 342.0

E* 435.0

E* 428.1

E* 310.1

B* 460.2

B* 424.1

B* 364.2

C* 420.2

C* 396.1

D* 456.2

D* 392.2

C* 358.1

B  394.1

B* 366.1

B* 414.2

C* 472.2

B* 372.1

D* 403.1

B* 388.1

B* 372.1

B* 358.1

B* 473.2

B* 436.0

B* 401.1

A  376.1

B* 373.1

B* 401.1

B* 416.1

B* 372.1

B* 473.2

B  604.2

C* 426.1

B* 373.1

D* 524.1

A* 424.1

D* 360.1

B* 420.1

B* 372.1

B* 468.2

B* 472.2

C* 432.0

D* 344.1

TABLE 3 STRUCTURE ACTIVITY (M + H)⁺

D* 521.2

C* 503.1

D* 456.1

D* 457.1

D* 485.1

D* 485.1

E* 399.1

D* 463.1

D* 427.1

E* 501.2

D* 456.1

D* 427.1

D* 485.1

D* 485.1

D* 457.1

D* 503.1

D* 463.1

D* 414.1

D 336.1

D 350.1

D 351.1

E* 288.1

E* 316.2

D* 362.2

D* 408.2

D* 344.2

D* 442.1

D* 372.2

D* 334.2

E* 315.2

D* 350.1

D* 394.1

E* 364.2

D* 400.2

D* 341.2

D* 346.2

D* 336.1

D* 358.2

D* 330.2

D* 364.2

D* 361.2

E* 346.2

E* 384.2

E* 350.1

D* 359.2

E* 334.2

E* 334.2

E* 346.2

E* 350.1

E* 384.2

E* 341.2

B* 340.1

C* 352.2

D* 409.1

D* 384.1

D* 491.1

E* 499.2

D* 497.1

E* 548.1

D* 518.1

D* 510.2

E* 499.2

D* 518.1

D* 428.1

D* 441.1

E* 497.1

D* 513.2

D* 484.1

D* 455.1

E* 554.2

D* 428.1

D* 427.1

E* 441.1

D* 497.1

D* 560.2

D* 441.1

D* 498.1

E* 436.1

E* 495.1

E* 510.2

D* 484.2

E* 427.1

E* 344.2

E* 418.1

D* 364.2

D* 418.2

B 576.2

C 456.2

E* 386.3

E* 386.3

D* 384.1

E* 395.1

D* 352.2

E* 418.1

D* 472.0

D* 384.1

D* 352.2

D* 384.1

D* 374.1

E* 358.2

D* 418.1

E* 370.1

B 349.1

B 393.1

C 333.1

B 383.1

B 351.1

C* 347.2

C* 407.1

D* 363.1

D* 363.1

C 377.1

D 391.2

E* 234.1

C* 404.2

C* 422.2

D* 296.1

D* 376.2

E* 282.1

E* 296.1

D* 338.1

E* 324.1

E* 324.1

D 336.1

D* 487.1

TABLE 4 ACTIV- STRUCTURE ITY (M + H)⁺

D* 322.1

E* 322.1

E* 348.2

E* 335.2

D* 408.1

TABLE 5 ABBREVI- NAME ATION STRUCTURE methyl Me —CH₃ ethyl Et —CH₂CH₃n-propyl nPr —CH₂CH₂CH₃ iso-propyl iPr —CH(CH₃)₂ n-butyl nBu—CH₂CH₂CH₂CH₃ tert-butyl tBu —C(CH₃)₃ sec-butyl —CH(CH₂)(CH₂CH₃)iso-butyl iBu —CH₂CH(CH₃)₂ n-pentyl nPent —CH₂CH₂CH₂CH₂CH₃ 3-pentanyl orpentan-3-yl —CH(CH₂CH₃)₂ amyl  CH₂CH(CH₃)CH₂CH₃ neopentyl —CH₂C(CH₃)₃3-methyl-2-butanyl —CH(CH₃)CH(CH₃)₂ tertiary amyl —C(CH₃)₂CH₂CH₃ n-hexylnHex —CH₂CH₂CH₂CH₂CH₂CH₃ phenyl Ph —C₆H₅ benzyl Bn —CH₂C₆H₅ acetyl Ac—C(═O)CH₃ tert-butyloxycarbonyl Boc —C(═O)OC(CH₃)₃tert-butyldimethylsilyl TBDMS —Si(Me)₂tBu para-methoxybenzyl PMB—CH₂C₆H₅(4-OMe) Biotin

Equivalents REFERENCES

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually incorporated by reference. In case ofconflict, the present application, including any definitions herein,will control.

EQUIVALENTS

While specific embodiments of the present disclosure have beendiscussed, the above specification is illustrative and not restrictive.Many variations of this disclosure will become apparent to those skilledin the art upon review of this specification. The full scope of thedisclosure should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in this specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present disclosure.

1-41. (canceled)
 42. A method of treating prostate cancer comprisingadministering to a subject a therapeutically effective amount of a FASNinhibitor.
 43. The method of claim 42, wherein the FASN inhibitor is acompound of formula (I):

or a pharmaceutically acceptable form thereof; wherein: R^(A) isselected from C₆₋₁₄ aryl and 5-14 membered heteroaryl; R^(B) is selectedfrom C₆₋₁₄ aryl and 5-14 membered heteroaryl; R^(C) is selected from—OH, —OR^(C1), —ON(R^(C2))₂, —N(R^(C2))₂, —C(═O)R^(C1), —CHO,—CO₂R^(C1), —C(═O)N(R^(C2))₂, —C(═NR^(C2))OR^(C1),—C(═NR^(C2))N(R^(C2))₂, —SO₂R^(C1), —S(═O)R^(C1), —Si(R^(C1))₃, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, with the proviso that R^(C) is not—CH₃; each instance of R^(C1) is, independently, selected from C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl; each instance of R^(C2) is,independently, selected from hydrogen, —OH, —OR^(C1), —N(R^(C3))₂, —CN,—C(═O)R^(C1), —C(═O)N(R^(C3))₂, —CO₂R^(C1), —SO₂R^(C1),—C(═NR^(C3))OR^(C1), —C(═NR^(C3))N(R^(C3))₂, —SO₂N(R^(C3))₂, —SO₂R^(C3),—SO₂OR^(C3), —SOR^(C1), —C(═S)N(R^(C3))₂, —C(═O)SR^(C3), —C(═S)SR^(C3),—P(═O)₂R^(C1), —P(═O)(R^(C1))₂, —P(═O)₂N(R^(C3))₂, —P(═O)(NR^(C3))₂,C₂₋₁₀ alkyl, C₂₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(C2) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring; or R^(B) and R^(C) together with the nitrogen (N) atom to whicheach is attached are joined to form a 5-14 membered ring; wherein: R^(B)is substituted with the group:-L-R^(D) wherein: L is a covalent bond or a divalent C₁₋₁₀ hydrocarbonchain, wherein one, two or three methylene units of L are optionally andindependently replaced with one or more —O—, —S—, —NR^(B8)—,—(C═NR^(B8))—, —C(═O)—, —C(═S)—, —S(═O)—, —S(═O)₂—, divalentcarbocyclyl, divalent heterocyclyl, divalent aryl or divalent heteroarylgroup; R^(D) is selected from —CN, —NO₂, —N₃, —SO₂H, —SO₃H,—C(═O)R^(B7), —CO₂H, —CHO, —C(OR^(B9))₂, —CO₂R^(B7), —OC(═O)R^(B7),—OCO₂R^(B7), —C(═O)N(R^(B8))₂, —OC(═O)N(R^(B8))₂, —NR^(B8)C(═O)R^(B7),—NR^(B8)CO₂R^(B7), —NR^(B8)C(═O)N(R^(B8))₂, —C(═NR^(B8))OR^(B7),—OC(═NR^(B8))R^(B7), —OC(═NR^(B8))OR^(B7), —C(═NR^(B8))N(R^(B8))₂,—OC(═NR^(B8))N(R^(B8))₂, —NR^(B8)C(═NR^(B8))N(R^(B8))₂,—C(═O)NR^(B8)SO₂R^(B7), —NR^(B8)SO₂R^(B7), —SO₂N(R^(B8))₂, —SO₂R^(B7),—SO₂OR^(B7), —OSO₂R^(B7), —S(═O)R^(B7), —OS(═O)R^(B7), —C(═S)N(R^(B8))₂,—C(═O)SR^(B7), —C(═S)SR^(B7), —SC(═S)SR^(B7), —P(═O)₂R^(B7),—OP(═O)₂R^(B7), —P(═O)(R^(B7))₂, —OP(═O)(R^(B7))₂, —OP(═O)(OR^(B9))₂,—P(═O)₂N(R^(B8))₂, —OP(═O)₂N(R^(B8))₂, —P(═O)(NR^(B8))₂,—OP(═O)(NR^(B8))₂, —NR^(B8)P(═O)(OR^(B9))₂, —NR^(B8)P(═O)(NR^(B8))₂,—B(OR^(B9))₂, —BR^(B7)(OR^(B9)), and tetrazolyl; each instance of R^(B7)is, independently, selected from C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl; each instance of R^(B8) is, independently, selected fromhydrogen, —OH, —OR^(B7), —N(R^(B9))₂, —CN, —C(═O)R^(B7),—C(═O)N(R^(B9))₂, —CO₂R^(B7), —SO₂R^(B7), —C(═NR^(B9))OR^(B7),—C(═NR^(B9))N(R^(B9))₂, —SO₂N(R^(B9))₂, —SO₂R^(B9), —SO₂OR^(B9),—SOR^(B7), —C(═S)N(R^(B9))₂, —C(═O)SR^(B9), —C(═S)SR^(B9),—P(═O)₂R^(B7), —P(═O)(R^(B7))₂, —P(═O)₂N(R^(B9))₂, —P(═O)(NR^(B9))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B8) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring; and each instance of R^(B9) is, independently, selected fromhydrogen, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B9)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring.
 44. The method of claim 43, wherein L is a covalentbond.
 45. The method of claim 44, wherein R^(D) is —CO₂H.
 46. The methodof claim 43, wherein R^(E) is selected from halogen, —OR^(B10) and—N(R^(B11))₂.
 47. The method of claim 43, wherein R^(C) is C₃₋₁₀ alkyl.48. The method of claim 43, wherein R^(C) is C₃₋₁₀ carbocyclyl.
 49. Themethod of claim 43, wherein R^(A) is C₆₋₁₄ aryl or 5-14 memberedheteroaryl, and R^(B) is C₆₋₁₄ aryl.
 50. The method of claim 49, whereinR^(A) is C₆₋₁₄ aryl and R^(B) is C₆₋₁₄ aryl.
 51. The method of claim 43,wherein R^(A) is 5-14 membered heteroaryl and R^(B) is C₆₋₁₄ aryl. 52.The method of claim 43, wherein the compound is of the formula (II):

or a pharmaceutically acceptable form thereof; wherein each group W—R¹,W—R², W—R³, W—R⁴, and W—R⁵ independently represents either a nitrogenatom (N) or C—R¹, C—R², C—R³, C—R⁴, or C—R⁵, respectively; and whereinR¹, R², R³, R⁴ and R⁵ are, independently, selected from the groupconsisting of hydrogen, halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH,—OR^(A1), —ON(R^(A2))₂, —N(R^(A2))₂, —N(OR^(A3))R^(A3), —SH, —SR^(A1),—SSR^(A3), —C(═O)R^(A1), —CO₂H, —CHO, —C(OR^(A3))₂, —CO₂R^(A1),—OC(═O)R^(A1), —OCO₂R A1, —C(═O)N(R_(A2))₂, —OC(═O)N(R^(A2))₂,—NR^(A2)C(═O)R^(A1), —NR^(A2)C₂R^(A1), —NR^(A2)C(═O)N(R^(A2))₂,—C(═NR^(A2))OR^(A1), —OC(═NR)R^(A1), —OC(═NR^(A2))OR^(A1),—C(═NR^(A2))N(R^(A2))₂, —OC(═NR^(A2))N(R^(A2))₂,—NR^(A2)C(═NR^(A2))N(R^(A2))₂, —C(═O)NR^(A2)SO₂R^(A1),—NR^(A2)SO₂R^(A1), —SO₂N(R^(A2))₂, —SO₂R^(A1), —SO₂OR^(A1), —OSO₂R^(A1),—S(═O)R^(A1), —OS(═O)R^(A1), —Si(R^(A1))₃,—OSi(R^(A1))₃—C(═S)N(R^(A2))₂, —C(═O)SR^(A1), —C(═S)SR^(A1),—SC(═S)SR^(A1), —P(═O)₂R^(A1), —OP(═O)₂R^(A1), —P(═O)(R^(A1))₂,—OP(═O)(R^(A1))₂, —OP(═O)(OR^(A3))₂, —P(═O)₂N(R^(A2))₂,—OP(═O)₂N(R^(A2))₂, —P(═O)(NR^(A2))₂, —OP(═O)(NR^(A3))₂,—NR^(A2)P(═O)(OR^(A3))₂, —NR^(A2)P(═O)(NR^(A2))₂, —P(R^(A3))₂,—P(R^(A3))₃, —OP(R^(A3))₂, —OP(R^(A3))₃, —B(OR^(A3))₂, or—BR^(A1)(OR^(A3)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl; or one or moreof R¹ and R², R² and R³, R³ and R⁴ or R⁴ and R⁵ are joined to form aC₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl or 5-14membered heteroaryl ring; each instance of R^(A1) is, independently,selected from C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl; each instance ofR^(A2) is, independently, selected from hydrogen, —OH, —OR^(A1),—N(R^(A3))₂, —CN, —C(═O)R^(A1), —C(═O)N(R^(A3))₂, —CO₂R^(A1),—SO₂R^(A1), —C(═NR^(A3))OR^(A1), —C(═NR^(A3))N(R^(A3))₂, —SO₂N(R^(A3))₂,—SO₂R^(A3), —SO₂OR^(A3), —SOR^(A1), —C(═S)N(R^(A3))₂, —C(═O)SR^(A3),—C(═S)SR^(A3), —P(═O)₂R^(A1), —P(═O)(R^(A1))₂, —P(═O)₂N(R^(A3))₂,—P(═O)(NR^(A3))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂-10 alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(A2)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring; and each instance of R^(A3) is, independently, selectedfrom hydrogen, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃-10 carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(A3)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring; wherein each group W—R⁶, W—R⁷, W—R⁸, W—R⁹, and W—R¹⁰independently represents either a nitrogen atom (N) or C—R⁶, C—R⁷, C—R⁸,C—R⁹, or C—R¹⁰, respectively; R⁶, R⁷, R⁸, R⁹ and R¹⁰ are, independently,selected from the group consisting of hydrogen, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OR^(B1), —ON(R^(B2))₂, —N(R^(B2))₂,—N(OR^(B3))R^(B3), —SH, —SR^(B1), —SSR^(B3), —C(═O)R^(B1), —CO₂H, —CHO,—C(OR^(B3))₂, —CO₂R^(B1), —OC(═O)R^(B1), —OCO₂R^(B1), —C(═O)N(R^(B2))₂,—OC(═O)N(R^(B2))₂, —NR^(B2)C(═O)R^(B1), —NR^(B2)CO₂R^(B1),—NR^(B2)C(═O)N(R^(B2))₂, —C(═NR^(B2))OR^(B1), —OC(═NR^(B2))R^(B1),—OC(═NR^(B2))OR^(B1), —C(═NR^(B2))N(R^(B2))₂, —OC(═NR^(B2))N(R^(B22),—NR^(B2)C(═NR^(B2))N(R^(B2))₂, —C(═O)NR^(B2)SO₂R^(B1),—NR^(B2)SO₂R^(B1), —SO₂N(R^(B2))₂, —SO₂R^(B1), —SO₂OR^(B1), —OSO₂R^(B1),—S(═O)R^(B1), —OS(═O)R^(B1), —Si(R^(B1))₃,—OSi(R^(B1))₃—C(═S)N(R_(B2))₂, —C(═O)SR^(B1), —C(═S)SR^(B1),—SC(S)SR^(B1), —P(═O)₂R^(B1), —OP(═O)₂R^(B1), —P(═O)(R^(B1))₂,—OP(═O)(R^(B1))₂, —OP(═O)(OR^(B3))₂, —P(═O)₂N(R^(B2))₂,—OP(═O)₂N(R^(B2))₂, —P(═O)(NR^(B2))₂, —OP(═O)(NR^(B2))₂,—NR^(B2)P(═O)(OR^(B3))₂, —R^(B2)P(═O)(NR^(B2))₂, —P(R^(B3))₂,—P(R^(B3))₃, —OP(R^(B3))₂, —OP(R^(B3))₃, —B(OR^(B3))₂,—BR^(B1)(OR^(B3)), C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, 5-14 membered heteroaryl, -L-R^(D) and —R^(E);or one or more of R⁶ and R⁷, R⁷ and R⁸, R⁸ and R⁹ or R⁹ and R¹⁰ arejoined to form a C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl or 5-14 membered heteroaryl ring; or R¹⁰ and R^(C) are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring;wherein at least one of R⁶, R⁷, R⁸, R⁹, and R¹⁰ is the group -L-R^(D);each instance of R^(B1) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl; each instance of R^(B2) is,independently, selected from hydrogen, —OH, —OR^(B1), —N(R^(B3))₂, —CN,—C(═O)R^(B1), —C(═O)N(R_(B3))₂, —CO₂R^(B1), —SO₂R^(B1),—C(═NR^(B3))OR^(B1), —C(═NR^(B3))N(R^(B3))₂, —SO₂N(R^(B3))₂, —SO₂R^(B3),—SO₂OR^(B3), —SOR^(B1), —C(═S)N(R_(B3))₂, —C(═O)SR^(B3), —C(═S)SR^(B3),—P(═O)₂R^(B1), —P(═O)(R^(B1))₂, —P(═O)₂N(R^(B3))₂, —P(═O)(NR^(B3))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B2) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring; each instance of R^(B3) is, independently, selected from hydrogen,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B3) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring; L is a covalent bond or a divalent C₁₋₁₀ hydrocarbon chain,wherein one, two or three methylene units of L are optionally andindependently replaced with one or more —O—, —S—, —NR^(B8)—,—(C═NR^(B8))—, —C(═O)—, —C(═S)—, —S(═O)—, —S(═O)₂—, divalentcarbocyclyl, divalent heterocyclyl, divalent aryl or divalent heteroarylgroup; and wherein R^(D) is selected from —CN, —NO₂, —N₃, —SO₂H, —SO₃H,—C(═O)R^(B7), —CO₂H, —CHO, —C(OR^(B9))₂, —CO₂R^(B7), —OC(═O)R^(B7),—OCO₂R^(B7), —C(═O)N(R^(B8))₂, —OC(═O)N(R^(B8))₂, —NR^(B8)C(═O)R^(B7),—NR^(B8)CO₂R^(B7), —NR^(B8)C(═O)N(R^(B8))₂, —C(═NR^(B8))OR^(B7),—OC(═NR^(B8))R^(B7), —OC(═NR^(B8))OR^(B7), —C(═NR^(B8))N(R^(B8))₂,—OC(═NR^(B8))N(R^(B8))₂, —NR^(B8)C(═NR^(B8))N(R^(B8))₂,—C(═O)NR^(B8)SO₂R^(B7), —NR^(B8)SO₂R^(B7), —SO₂N(R^(B8))₂, —SO₂R^(B7),—SO₂OR^(B7), —OSO₂R^(B7), —S(═O)R^(B7), —OS(═O)R^(B7), —C(═S)N(R^(B8))₂,—C(═O)SR^(B7), —C(═S)SR^(B7), —SC(═S)SR^(B7), —P(═O)₂R^(B7),—OP(═O)₂R^(B7), —P(═O)(R^(B7))₂, —OP(═O)(R^(B7))₂, —OP(═O)(OR^(B9))₂,—P(═O)₂N(R^(B8))₂, —OP(═O)₂N(R^(B8))₂, —P(═O)(NR^(B8))₂,—OP(═O)(NR^(B8))₂, —NR^(B8)P(═O)(OR^(B9))₂, —NR^(B8)P(═O)(NR^(B8))₂,—B(OR^(B9))₂, —BR^(B7)(OR^(B9)) and tetrazolyl; each instance of R^(B7)is, independently, selected from C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl; each instance of R^(B8) is, independently, selected fromhydrogen, —OH, —OR^(B7), —N(R^(B9))₂, —CN, —C(═O)R^(B7),—C(═O)N(R^(B9))₂, —CO₂R^(B7), —SO₂R^(B7), —C(═NR^(B9))OR^(B7),—C(═NR^(B9))N(R^(B9))₂, —SO₂N(R^(B9))₂, —SO₂R^(B9), —SO₂OR^(B9),—SOR^(B7), —C(═S)N(R^(B9))₂, —C(═O)SR^(B9), —C(═S)SR^(B9),—P(═O)₂R^(B7), —P(═O)(R^(B7))₂, —P(═O)₂N(R^(B9))₂, —P(═O)(NR^(B9))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B8) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring; and each instance of R^(B9) is, independently, selected fromhydrogen, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B9)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring; R^(E) is selected from halogen, —OH, —OR^(B10),—ON(R^(B11))₂, —N(R^(B11))₂, —N(OR^(B12))R^(B12), —SH, —SR^(B10),—SSR^(B12), —OC(═O)R^(B10), —OCO₂R^(B10), —OC(═O)N(R^(B1))₂,—NR^(B11)C(═O)R^(B10), —NR^(B11)CO₂R^(B10), —NR^(B11)C(═O)N(R^(B11))₂,—OC(═NR^(B11))R^(B11), —OC(═NR^(B11))OR^(B10),—OC(═NR^(B11))N(R^(B11))₂, —R^(B11)C(═NR^(B11))N(R^(B11))₂,—R^(B11)SO₂R^(B10), —OSO₂R^(B10), —OS(═O)R^(B10), —Si(R^(B10))₃,—OSi(R^(B10))₃, —SC(S)SR^(B10), —OP(═O)₂R^(B10), —OP(═O)(R^(B10))₂,—OP(═O)(OR^(B12))₂, —OP(═O)₂N(R^(B11))₂, —OP(═O)(NR^(B11))₂,—NR^(B11)P(═O)(OR^(B12))₂, —NR^(B11)P(═O)(NR^(B11))₂, —P(R^(B12))₂,—P(R^(B12))₃, —OP(R^(B12))₂, —OP(R^(B12))₃, 3-14 membered heterocyclyland 5-14 membered heteroaryl, wherein the point of attachment of the3-14 membered heterocyclyl or 5-14 membered heteroaryl group is on anitrogen atom; each instance of R^(B10) is, independently, selected fromC₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl; each instance of R^(B11) is,independently, selected from hydrogen, —OH, —OR^(B10), —N(R^(B12))₂,—CN, —C(═O)R^(B10), —C(═O)N(R^(B12))₂, —CO₂R^(B10), —SO₂R^(B10),—C(═NR^(B12))OR^(B10), —C(═NR^(B12))N(R^(B12))₂, —SO₂N(R^(B12))₂,—SO₂R^(B12), —SO₂OR^(B12), —SOR^(B10), —C(═S)N(R^(B12))₂,—C(═O)SR^(B12), —C(═S)SR^(B12), —P(═O)₂R^(B10), —P(═O)(R^(B10))₂,—P(═O)₂N(R^(B12))₂, —P(═O)(NR^(B12))₂, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl,C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(B11) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring; each instance of R^(B12)is, independently, selected from hydrogen, C₁₋₁₀ alkyl, C₁₋₁₀perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, or two R^(B12) groups are joined toform a 3-14 membered heterocyclyl or 5-14 membered heteroaryl ring;R^(C) is selected from —OH, —OR^(C1), —ON(R^(C2))₂, —N(R^(C2))₂,—C(═O)R^(C1), —CHO, —CO₂R^(C1), —C(═O)N(R^(C2))₂, —C(═NR^(C2))OR^(C1),—C(═NR^(C2))N(R^(C2))₂, —SO₂R^(C1), —S(═O)R^(C1), —Si(R^(C1))₃,C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, with the proviso that R^(C) isnot —CH₃; each instance of R^(C1) is, independently, selected from C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl; and each instance of R^(C2) is,independently, selected from hydrogen, —OH, —OR^(C1), —N(R^(C3))₂, —CN,—C(═O)R^(Cl), —C(═O)N(R^(C3))₂, —CO₂R^(C1), —SO₂R^(C1),—C(═NR^(C3))OR^(C1), —C(═NR^(C3))N(R^(C3))₂, —SO₂N(R^(C3))₂, —SO₂R^(C3),—SO₂OR^(C3), —SOR^(C1), —C(═S)N(R^(C3))₂, —C(═O)SR^(C3), —C(═S)SR^(C3),—P(═O)₂R^(C1), —P(═O)(R^(C1))₂, —P(═O)₂N(R^(C3))₂, —P(═O)(NR^(C3))₂,C₂₋₁₀ alkyl, C₂₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(C2) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring.
 53. The method of claim 52, wherein at least one of R⁶, R⁷, R⁸, R⁹and R¹⁰ is the group —R^(E).
 54. The method of claim 42, wherein theFASN inhibitor is:

or a pharmaceutically acceptable form thereof.
 55. The method of claim42, which further comprises administration of one or more anti-canceragents.
 56. The method of claim 55, wherein the anti-cancer agent isselected from an interferon, an antibody, an anti-estrogen, an LHRHagonist, an anti-androgen, a photodynamic therapy, phthalocyanine,photosensitizer Pc4, demethoxy-hypocrellin A, a nitrogen mustard, anitrosourea, an alkylsulphonate, a triazene, a platinum containingcompound, a vinca alkaloid, a taxoid, an epipodophyllin, ananti-metabolite, a DHFR inhibitor, an IMP dehydrogenase inhibitor, aribonuclotide reductase inhibitor, a uracil analog, a cytosine analog, apurine analog, a vitamin D3 analog, an isoprenylation inhibitor, adopaminergic neurotoxin, a cell cycle inhibitor, actinomycin, bleomycin,anthracycline, MDR inhibitor, Ca2+ ATPase inhibitor, and a tyrosinekinase inhibitor.
 57. A method of inhibiting fatty acid synthase in asubject having prostate cancer, where in the method comprisesadministering to the subject a therapeutically effective amount of afatty acid synthase inhibitor.
 58. The method of claim 57, wherein theFASN inhibitor is a compound of formula (I):

or a pharmaceutically acceptable form thereof; wherein: R^(A) isselected from C₆₋₁₄ aryl and 5-14 membered heteroaryl; R^(B) is selectedfrom C₆₋₁₄ aryl and 5-14 membered heteroaryl; R^(C) is selected from—OH, —OR^(C1), —ON(R^(C2))₂, —N(R^(C2))₂, —C(═O)R^(C1), —CHO,—CO₂R^(C1), —C(═O)N(R^(C2))₂, —C(═NR^(C2))OR^(C1),—C(═NR^(C2))N(R^(C2))₂, —SO₂R^(C1), —S(═O)R^(C1), —Si(R^(C1))₃, C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl, with the proviso that R^(C) is not—CH₃; each instance of R^(C1) is, independently, selected from C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14 memberedheteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄aryl, and 5-14 membered heteroaryl; each instance of R^(C2) is,independently, selected from hydrogen, —OH, —OR^(C1), —N(R^(C3))₂, —CN,—C(═O)R^(C1), —C(═O)N(R^(C3))₂, —CO₂R^(C1), —SO₂R^(C1),—C(═NR^(C3))OR^(C1), —C(═NR^(C3))N(R^(C3))₂, —SO₂N(R^(C3))₂, —SO₂R^(C3),—SO₂OR^(C3), —SOR^(C1), —C(═S)N(R^(C3))₂, —C(═O)SR^(C3), —C(═S)SR^(C3),—P(═O)₂R^(C1), —P(═O)(R^(C1))₂, —P(═O)₂N(R^(C3))₂, —P(═O)(NR^(C3))₂,C₂₋₁₀ alkyl, C₂₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(C2) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring; or R^(B) and R^(C) together with the nitrogen (N) atom to whicheach is attached are joined to form a 5-14 membered ring; wherein: R^(B)is substituted with the group:-L-R^(D) wherein: L is a covalent bond or a divalent C₁₋₁₀ hydrocarbonchain, wherein one, two or three methylene units of L are optionally andindependently replaced with one or more —O—, —S—, —NR^(B8)—,—(C═NR^(B8))—, —C(═O)—, —C(═S)—, —S(═O)—, —S(═O)₂—, divalentcarbocyclyl, divalent heterocyclyl, divalent aryl or divalent heteroarylgroup; R^(D) is selected from —CN, —NO₂, —N₃, —SO₂H, —SO₃H,—C(═O)R^(B7), —CO₂H, —CHO, —C(OR^(B9))₂, —CO₂R^(B7), —OC(═O)R^(B7),—OCO₂R^(B7), —C(═O)N(R^(B8))₂, —OC(═O)N(R^(B8))₂, —NR^(B8)C(═O)R^(B7),—NR^(B8)CO₂R^(B7), —NR^(B8)C(═O)N(R^(B8))₂, —C(═NR^(B8))OR^(B7),—OC(═NR^(B8))R^(B7), —OC(═NR^(B8))OR^(B7), —C(═NR^(B8))N(R^(B8))₂,—OC(═NR^(B8))N(R^(B8))₂, —NR^(B8)C(═NR^(B8))N(R^(B8))₂,—C(═O)NR^(B8)SO₂R^(B7), —NR^(B8)SO₂R^(B7), —SO₂N(R^(B8))₂, —SO₂R^(B7),—SO₂OR^(B7), —OSO₂R^(B7), —S(═O)R^(B7), —OS(═O)R^(B7), —C(═S)N(R^(B8))₂,—C(═O)SR^(B7), —C(═S)SR^(B7), —SC(═S)SR^(B7), —P(═O)₂R^(B7),—OP(═O)₂R^(B7), —P(═O)(R^(B7))₂, —OP(═O)(R^(B7))₂, —OP(═O)(OR^(B9))₂,—P(═O)₂N(R^(B8))₂, —OP(═O)₂N(R^(B8))₂, —P(═O)(NR^(B8))₂,—OP(═O)(NR^(B8))₂, —NR^(B8)P(═O)(OR^(B9))₂, —NR^(B8)P(═O)(NR^(B8))₂,—B(OR^(B9))₂, —BR^(B7)(OR^(B9)), and tetrazolyl; each instance of R^(B7)is, independently, selected from C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀alkenyl, C₂₋₁₀ alkynyl, 3-14 membered heteroaliphatic, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl; each instance of R^(B8) is, independently, selected fromhydrogen, —OH, —OR^(B7), —N(R^(B9))₂, —CN, —C(═O)R^(B7),—C(═O)N(R^(B9))₂, —CO₂R^(B7), —SO₂R^(B7), —C(═NR^(B9))OR^(B7),—C(═NR^(B9))N(R^(B9))₂, —SO₂N(R^(B9))₂, —SO₂R^(B9), —SO₂OR^(B9),—SOR^(B7), —C(═S)N(R^(B9))₂, —C(═O)SR^(B9), —C(═S)SR^(B9),—P(═O)₂R^(B7), —P(═O)(R^(B7))₂, —P(═O)₂N(R^(B9))₂, —P(═O)(NR^(B9))₂,C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, 3-14membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl,C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B8) groups arejoined to form a 3-14 membered heterocyclyl or 5-14 membered heteroarylring; and each instance of R^(B9) is, independently, selected fromhydrogen, C₁₋₁₀ alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,3-14 membered heteroaliphatic, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(B9)groups are joined to form a 3-14 membered heterocyclyl or 5-14 memberedheteroaryl ring.
 59. The method of claim 57, wherein the FASN inhibitoris:

or a pharmaceutically acceptable form thereof.
 60. The method of claim57, which further comprises administration of one or more anti-canceragents.
 61. The method of claim 60, wherein the anti-cancer agent isselected from an interferon, an antibody, an anti-estrogen, an LHRHagonist, an anti-androgen, a photodynamic therapy, phthalocyanine,photosensitizer Pc4, demethoxy-hypocrellin A, a nitrogen mustard, anitrosourea, an alkylsulphonate, a triazene, a platinum containingcompound, a vinca alkaloid, a taxoid, an epipodophyllin, ananti-metabolite, a DHFR inhibitor, an IMP dehydrogenase inhibitor, aribonuclotide reductase inhibitor, a uracil analog, a cytosine analog, apurine analog, a vitamin D3 analog, an isoprenylation inhibitor, adopaminergic neurotoxin, a cell cycle inhibitor, actinomycin, bleomycin,anthracycline, MDR inhibitor, Ca2+ ATPase inhibitor, and a tyrosinekinase inhibitor.
 62. A method of treating a cancer comprisingadministering to a subject a therapeutically effective amount of

or a pharmaceutically acceptable form thereof.
 63. The method of claim62, wherein the cancer is selected from bladder cancer, brain cancer,breast cancer, colorectal cancer, esophageal cancer, endometrial cancer,gastric cancer, gastrointestinal stromal tumor, kidney cancer, livercancer, lung cancer, mesothelioma, multiple myeloma, neuroblastoma, oralcancer, ovarian cancer, pancreatic cancer, prostate cancer, Paget'sdisease of the vulva, retinoblastoma, soft tissue sarcoma, skin canceror thyroid cancer.
 64. The method of claim 62, wherein the cancer isprostate cancer.
 65. The method of claim 62, wherein the cancer is livercancer.
 66. The method of claim 62, wherein the cancer is colorectalcancer.
 67. The method of claim 62, wherein the cancer is breast cancer.68. The method of claim 62, wherein the cancer is ovarian cancer. 69.The method of claim 62, wherein the cancer is hepatocellular carcinoma.70. The method of claim 62, which further comprises administration ofone or more anti-cancer agents.
 71. The method of claim 70, wherein theanti-cancer agent is selected from an interferon, an antibody, ananti-estrogen, an LHRH agonist, an anti-androgen, a photodynamictherapy, phthalocyanine, photosensitizer Pc4, demethoxy-hypocrellin A, anitrogen mustard, a nitrosourea, an alkylsulphonate, a triazene, aplatinum containing compound, a vinca alkaloid, a taxoid, anepipodophyllin, an anti-metabolite, a DHFR inhibitor, an IMPdehydrogenase inhibitor, a ribonuclotide reductase inhibitor, a uracilanalog, a cytosine analog, a purine analog, a vitamin D3 analog, anisoprenylation inhibitor, a dopaminergic neurotoxin, a cell cycleinhibitor, actinomycin, bleomycin, anthracycline, MDR inhibitor, Ca2+ATPase inhibitor, and a tyrosine kinase inhibitor.
 72. A method oftreating an obesity related disorder comprising administering to asubject a therapeutically effective amount of

or a pharmaceutically acceptable form thereof.
 73. The method of claim72, wherein the obesity related disorder is selected from Type IIdiabetes mellitus, elevated blood pressure, elevated cholesterol levels,ischemic heart disease, arterial vascular disease, angina, myocardialinfarction, stroke, migraines, congestive heart failure, deep veinthrombosis, pulmonary embolism, gall stones, gastroesophagael refluxdisease, obstructive sleep apnea, obesity hypoventilation syndrome,asthma, gout, poor mobility, back pain, erectile dysfunction, urinaryincontinence, liver injury, fatty liver, and chronic renal failure.